Document pBgQjv88Z8X6wjKpYR6qzv5Rw

3M Environmental Laboratory FinalReport-AnalyticaSltudy Single-dosIentravenouPsharmacokineticStudy ofT-6246inRabbits In-VivoStudy ReferenceNumber: HWI#6329-159 Study Number: AMDT-042095.1 TestSubstance: FC-95 (T-6246) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,NIN 55106 Name and Address ofTestingFacility: 3M EnvironmentalTechnology & Services 935 Bush Avenue St.Paul,MN 55106 Method Numbers AMDT-M-1-0, AMDT-M-2-0, AMDT-M4-0, AMDT-M-8-0, AMDT-M-14-0, and Revisions: Thermal Extractioonf Fluorideby means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver FluorideMeasurement by Means ofan OrionEA940 ExpandableIon Analyzer ExtractionofFluorochemicalsfrom RabbitLiver AnalysisofFluorideUsing theSkalarSegmented Flow Analyzer with Ion SelectivEelectrode Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Serum InitiatioDnate: See attachedprotocol Author:James D. Johnson A dB es D. John 0 a dy Directo& Completion Date 000(','Oll 1.0 SUMMARY Rabbitserum and liverwere analyzedforfluorinecontentatvarioustimesafter rabbitswere dosed intravenouslwyith a singledose ofT-6246,a 0.12% sol'utionof FC-95. Doses areexpressedasFC-95 equivalentsT.he fluorineinserum was detectedaftera singledose of0.12 mg/kg and after0.60 mg/kg, but not aftera 0.012 mg/kg dose at28 days postdose.The method isnot sensitivenough to detect and measure fluoridaefterthe0.012 mg/kg dose. The half-lifoef perfluorooctanesulfonaatneionwas found tobe one month or greaterinserum and liver. The averageliverfluoridieonconcentratioinnGroup 5 animals(0.60mg/kg),at day 14 postdose was 1.48ppm and forfourotheranimalsinthe same group sacrificeadtday 28 theconcentratiownas 1.23ppm. Thus, thedecreasein concentrationinliverfrom 14 days to28 dayspost dose isslow;based on justthese two pointsthehalf-lifiesgreaterthan 1 month. 2.0 INTRODUCTION This studywas designedtoprovideinformationastowhetherthe perfluorooctanesulfonaatneiondoes go totheliverand othertissueswhen the materialisadministeredin an intravenousdose,and to ascertaitnhechange in concentratiownith time afterdose inserum and liver. Itisknown from studiesdone previouslywithratsthatthehalf-lifoef perfluorooctanesulfonaantieonisquitelong(>1 month). Itwas expectedthatthe half-lifienrabbitswould alsobe long.Perfluorooctanesulfonantieonisa likely biotransforrnatipornoduct of severalfluorochemicaltshataretobe testedfor dermal absorptioninotherstudies.The pharmacokineticsoftheperfluorooctanesulfonateanion isvery relevanto thesedennal absorptionstudies. 3.0 TEST MATERIALS 3.1Test,Control,and ReferenceSubstancesand Matrices 3.1.1AnalyticalReferenceSubstance:FC-95,lot161 or 171.They are equivalent. 3.1.2AnalyticalReferenceMatrix: Bovine liverand bovineserum. 3.1.3AnalyticalControlSubstance: None 3.1.4AnalyticalControlMatrix: Bovine liverand bovineserum 0 0 0 (i O'A-v2 3.2SourceofMaterial3sM: ICP/PCPDivisioFnorFC-95.Bovinelivefrrom grocerystore,bovine serum from Sigma Chemical Company 3.3.Purity and Strength of Reference Substance: Responsibilityof Sponsor. 3.4 Stabilityof Reference Substance: To be determined by Sponsor. 3.5 Storage Conditions for Test Materials:Room temperatureforFC-95. For biologicalsamples,the storageis-201 00C. 3.6 Dispositionof Specimens: Biologicaltissuesand fluidswillbe retainedper GLP Regulationforthetime periodrequiredforstudieslongerthan 28 days. 4.0 EXPERIMENTAL-Overview Serum and tissuesfrom animals dosed as described(HWI#6329-159), were available foranalysisforfluorinecompounds. Sinceperfluorooctanesulfonataenion isnot biotransfonned,the analysiswas accomplished with combustion and subsequent analysisforfluorine.The fluorinedataarerelatedirectlyto perfluorooctanesulfonateconcentrationT.he fluorineanalysisof serum collectedatdifferentime intervalasfterdosingprovidesdatawhich can be interpretepdharmacokinetically. Additionalanalysisof liversamples providesevidencethattheperfluorooctanesulfonateanion isstilplresentat28 days. 5.0 EXPERIMENTAL -Methods 5.1 ANMT-M-1-0, Thermal Extractionof Fluorideby Means' of a Modified Dohrmann DX2000 Organic HalideAnalyzer-Liver 5.2AAMT-M-2-0, FluorideMeasurem entby Means of an Orion EA940 Expandable Ion Analyzer 5.3 AMDT-M4-0. Extractionof Fluorochemicalsfrom RabbitLiver 5.4 AMDT-M-8-0, AnalysisofFluorideUsing theSkalarSegmented Flow Analyzer with Ion SelectiveElectrode 5.5 AMDT-M-14-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum 0()0()103 3 6.0DATA ANALYSIS Raw dataareshowninTableI (Appendi9x.11)D.osesareadjustefdortheFC-95 equivalents.The administereddose was T-6246 which isa watersolutionof0.12% FC-95. Serum concentrationasreobviouslynotdifferentthanthecontrol concentrationfsorthe0.006and 0.012mgikg doses.The concentratioinnserum is plottedforGroup 4 and Group 5 (seepages 140-141).For Group 5 (0.60mg/kg dose),iftheaverageconcentratioinsfittoa leastsquareregressioYn=ae', r'=0.95,a--l.62k,---l.099x 10-',k--0.693overtl,2a,nd tl,2=2d6ays. Similarly, t,,=48days forGroup 4. Thus,ifPerfluorooctanesulfoniaotneisformed from biotransformatioan,largeportionoftheamount formed willbe presentintheserum at28 days. The averageliverfluorideion concentratioinnGroup 5 animals,atday 14 postdose was 1.48ppm and forfourotheranimalsinthesame group sacrificeadtday 28 the concentratiownas 1.23ppm. Thus,thedecreaseinconcentratioinnliverfrom 14 days to28 days postdose isslow;based onjustthesetwo pointsthehalf-lifies greaterthan 1 month. Overall,foralltime periodsthe serum and liverlevelsareas expectedforthe intravenousdose. The abilittyodetectlevelsinliverafteran intravenousdose of 120 ug/kg suggeststhatwhen perfluorooctanesulfonaitsethemarker,dermal absorptionstudieswillbe quitesensitive. 6.1 Circumstances That May Affectthe Quality ofthe Data: These valuesfor concentratioanrefrom combustionanalysesofthebiologicamlaterial.The recoveriesarebased on spikingthebiologicamlaterialwithknown amounts of FC-95. The fluorineismeasured by selectivieonelectrodaend theOrion meter is calibrateodver a 5 pointrange.However, ifthereisa differenrtesponseatdifferent concentrationosfFC-95 otherthanthepointused asa calibratiocnheck,therecould be a biasatthoseconcentrationesven though theamount of fluorineisbeing measured accurately.This can come from othervariablesinthemethod such as the efficiencoyf combustion.Thus,thevaluesreportedforconcentratioanrenot absolutevalues.The valuesarecertainlsyufficientto make thepointthatthe perfluorooctanesulfonaatneionispresentat28 days inliverand serum,thatthe halflifeison the orderof a month orlonger,and thatthemethod can detect perfluorooctanesulfonaitneserum aftera dose somewhere between 0.012 and 0.12 mglkg. This method allowssufficienstensitiviftoyrdermalabsorptionstudies where some ofthe appliedfluorochemicalisbiotransformedtoperfluorooctanesulfonateanion. 7.0CONCLUSION The half-loifpeerfluorooctanesulifnolniavteaerndserumaftearnintravenous dose isone month or greaterinrabbits.The perfluorooctanesulfonaatneioncan be detectedby thecombustion method and subsequentfluorideanalysisat28 days after eithera 0.12or a 0.60 mg/kg intravenousdose. The 0.012 mg/kg intravenousdose doesnotprovidesufficienftluoridieonforthismethod. The method would probablybe ableto detecta dose somewhere between 0.012 and 0.12 mg/kg. Analysisof fluorineat28 days inliverand serum providesa marker fordermal absorptionfordermallyappliedfluorochemicaltshatarebiotransfonnedto" perfluorooctanesulfonaatneion. 8.0 MAINTENANCE OF RAW DATA AND RECORDS 8.1 Raw Data and Data: Raw data,approved protocol,approved finalreport, appropriatsepecimens,and electronidcatawillbe maintainedinthe AMDT Archives. 9.0 APPENDICES 9.1 Protocoland Amendments 9.1.1Protocoland FinalReport:HWI#6329-159 "SingleDose Intravenous PharmacokineticStudy ofT-6246 inRabbits" (ProtocoltypeTP8084.PK for dosing of animals,tissuecollectione,tc.) 9.1.2 AnalyticaplrotocolAMDT-042095.1 9.2 Signed Reports from IndividualScientistsN:one 9.3 QualityAssurance Unit Statement:See attached 9.4 Key PersonnelInvolvedintheStudy: See attached 9.5 Materialsand Equipment: See methods 9.6 Solutions,Reagents,and Standards:See methods 9.7 Sample Preparation:See methods 9.8 QualityControl Practices:See methods 0()0()0.1 5 9.9TestMethods:SeeProtocoAlMDT-042095.1 9.10InstrumenStettingSse:emethods '9.11Data:Seeattached: 9.11.S1ummaryandrawdata;ug F-inwholelivearsdeterminbeyd thermal extractiofnollowedby analysiussingOrion ionanalyzer. 9.11.2Summary and raw data;ug F- inwhole liveras determinedby thermal extractiofnollowedby analysiussingSkalarsegmented flow analyzerwith ion selectivelectrode. 9.11.3Summary and raw data;ppm F- in serum as determined by thermal extractiofnollowedby analysiussingOrion ionanalyzer. 9.11.4Summary and raw data;ppm F-inserum as determinedby thdnnal extractiofnollowedby analysiussingSkalarsegmented flow analyzerwith ion selectivelectrode. 000()OC3 6 9.1.1Protocoland FinalReport:HWI#6329-159 "Single Dose IntravenousPhannacoldneticStudy ofT-6246 in Rabbits" (ProtocoltypeTP8084.PK fordosingof animals, tissuecollectione,tc.) 0()0007 HAZLUN:C:N W IS C 0 N S IN P(DST OFFIC-' EOX 75,45 Nil-AOISOtl,kj','5!3707-75.-45 SRonsor: 3M St. Paul, Minnesota FINAL REPORT a CORNING Cortipany -C SEP 14 1995 31"A Study Title: Singl6-Dose IntravenousPhamacokinetic Study of T-6246 in Rabbits Author: Steven M. Glaza Study Completion Date: September 14, 1995 Performing--Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-159 P h o n e 608-241-4471 E X P R E S S -PvlA 1 0 E L IV E R Y: Page I of 38 330 1 K IN S M A N BLVD. Fax MADISON, 0 ('01 6 0 8 -2 4 1 - 7 2 2 7 W1 53704 Page 2 of 38 HWI 6329-159 QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality Assurance Unit of Hazleton Wisconsin, Inc., in accordancewith the Food and Drug Administration (FDA)Good LaboratoryPracticeRegulations,21 CFR 58.35 (b) (6) (7). The following inspectionswere conducted and findings reported to*the Study Director and management. Written status reports of inspections and findings are issued to Hazleton management monthly according to standard operating procedures. Inspection Dates From To Phase Date Reported to Date to Study Director Management 03/22/95 04/10/95 04/27/95 OY/05/95 09/12/95 03/22/95 04/10/95 04/27/95 07/10/95 09/12/95 Protocol Review Protocol Amendment Animal Observation Data/Report Review Report Rereview 03/22/95 04/10/95 04/27/95 07/10/95 09/12/95 03/10/95 05/10/95 05/10/95 08/10/95 10/10/95 Represefitit-lvQed,a-lity Assurance Unit Date Page 3 of 38 STUDY IDENTIFICATION Single-Dose Intravenous Pharmacokinetic Study of T-6246 in Rabbits HWI 6329-i59 Test Material T-6246 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Sponsor's Representative John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Study Director Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, Wl 53707-7545 (608) 241-7292 Study Location Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, WI 53704 Study Timetable Study InitiationDate Experimental(In-life)Start Date In-life End Date Experimental Termination Date Study Completion Date March 30, 1995 April 4, 1995 May 1, 1995 September 14, 1995 September 14, 1995 ()00()ILO Page 4 of 38 KEY PERSONNEL Acute Toxicology Steven M. Glaza Study Director Manager Francis (Bud) W. McDonald Study Coordinator Patricia Padgham In-life Supervisor Rose M. Bridge Report Supervisor Toxicology Support Kathy Myers Manager Calvin L. Horton Supervisor HWI 6329-159 Quality Assurance Sherry R. W. Petsel Manager Laboratory Animal Medicine Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Anatomical Pathology Thomas E. Palmer, PhD Anatomical Pathologist Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data o(JO011 Page 5 of 38 CONTENTS Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compliance Test and Control Materials Test System Procedures Results Discussion Signature Pathology Report Table 1 Individual Body Weights (g) 2 Individual Clinical Signs 3 Individual Pathology Comments 4 Individual Animal Tissue Weights and Bile Volumes Appendix A Protocol Deviations Protocol TP8084.PK Protocol Amendment No. 1 HWI 6329-159 Paqe 2 3 4 6 8 8 8 9 10 13 13 14 15 16 18 21 22 25 26 27 37 ooocILA'. Page 6 of 38 SUMMARY HWI 63291-159 This study was done to assess the level of systemic exposure of T-6246 when administeredby a single intravenousinjectionto rabbits. The study was conducted using four male and four female acclimated rabbits of the Hra:(NZW)SPF strain for each treatment group as follows: GrouD Dose Level Number of Animals Test Material (mg/kq) Males Females 1 (Control) Sterile water 0 4 4 2 (Low) T-6246 5 4 4 3 (Medium) T-6246 10 4 4 4 (High) T-6246 100 4* 4 5 (High) T-6246 Soo 4* 4 one animal sacrificed after treatment due to a possible broken back and replaced with another male animal. The animals received a single intravenousinjection of the test material at the indicated dose level into the marginal ear vein of the right ear. The dose volume was 0.5 mL/kg of body weight for Groups 1 through 4 and was 0.75 mL/kg for Group 5. Two animals/sex/doselevel were sacrificed on Day 15 and the remaining animals (two animals/sex/dose level) were sacrificed on Day 28. Clinical observations were conducted predose and at approximately 0.5, 2, and 4 hours after intravenousinjection. Additional clinical observationsand twice a day mortality checks were conducted daily thereafter until the scheduled sacrifice interval (Day 15 or Day 28). Body weights were determined on Day -7 for randomization purposes, before test or control material administration(Day 1), and at the scheduled sacrificeinterval (Day 15 or Day 28). A blood sample (approximately4 mL) was collected from a marginal ear vein (left ear) of the animals at 4-, 8-, 12-, 24-, and 48-hours post-injection, and on Day S. An approximate 4-mL blood sample was also collected on Days 15 and 22 for the animals scheduled for sacrifice on Day 28. In addition, at the time of the scheduled sacrifice (Day 15 or Day 28), approximately 20 mL of blood was obtained from each animal. All samples were centrifuged, separated into serum and cellular fractions, and sent to the Sponsor. On Day 15 or 28, the animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, and exsanguinated. An abbreviated gross necropsy examination was not done, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected, weighed (volume only determined for bile), and sent frozen to the Sponsor. o o0 Page 7 of 38 HWI 6329-159 Intravenousinjectionof T-6246did not result in any test material-related changes in body weight gain. All animalsappearedclinicallynormal throughout the study with the exception of one Group 4 and one Group 5 male animals that were sacrificedon Days 2 and 1, respectively,due to injury (possible broken backs). These animals were replacedand the replacement animals appearedclinically normal throughoutthe study. 0 0 (11 Page 8 of 38 HWI 6329-159 OBJECTIVE The objective of this study was to assess the level of systemic exposure to the test material, T-6246, when administered as a single intravenous injection to rabbits. REGULATORY COMPLIANCE This study was conducted in accordance with the U.S. Food and Drug Administration's Good Laboratory Practice Regulations for Nonclinical Laboratory Studies, 21 CFR 58, with the exception that analysis of the test mixtures for concentration,homogeneity/solubility,and stabilitywas not conducted. All procedures used in this study were in compliance with the Animal Welfare Act Re@ulations. In the opinion of the Sponsor and study director, the study did not unnecessarily duplicate any previous work. TEST AND CONTROL MATERIALS Identification The test material was identified as T-6246 and described as a clear, colorless liquid. The control material was Sterile Water for Injection,USP (Abbott Laboratories, Lot No. 86-748-DM-02; Exp. April 1, 1996), and was described as a clear, colorlessliquid. Purity and Stability The Sponsor assumes responsibilityfor test material purity and stability determinations(includingunder test conditions). Analysis of the test material mixtures for concentration,homogeneity/solubility,and stability was not conducted or requested by the Sponsor. The purity and stability of the control material were considered to be adequate for the purposes of this study. Storage and Retention The test material was.stored at room temperature. The control material was stored refrigerated. Any unused test material was returned to the Sponsor after completion of all in-life testing accordingto Hazleton Wisconsin (HWI) Standard Operating Procedure (SOP). Any remaining vehicle may be used for other testing and will not be discarded after issuance of the final report. ooooIL5 Page 9 of 38 Safety Precautions HWI 6329-159 The test and control material handlingprocedureswere according to HWI SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPFstrain were received from HRP,'Inc., Kalamazoo, Michigan on March 8, 1995 and maintained at the Hazleton Wisconsin facility at 3301 Kinsman Boulevard,Madison, Wisconsin. Housing After receipt,the animalswere acclimatedfor a period of at least 7 days. During acclimationand throughoutthe study, the animals were individually housed in screen-bottomstainlesssteel cages in temperature-and humiditycontrolledquarters. Environmentalcontrols for the animal room were set to maintain a temperatureof 19' to 23*C, a relativehumidity of 5m. 20%, and a 12-hourlight/12-hourdark lightingcycle. In cases where variationsfrom these conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal Diet The animals were provided access to water ad 7ibitum and a measured amount of LaboratoryRabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturerfor nutritionalcomponents and environmental contaminants. Samples of the water are periodicallyanalyzed by HWI. There were no known contaminantsin the feed or water at levels that would have interferedwith or affectedthe resultsof the study. Selection of Test Animals The animals were identifiedby animal number and correspondingear tag and were placed into study groups using a stratifiedbody weight randomization program. The randomizationbody weights were determined on Day -7. The weight variation of the animals for each group of each sex selected for the study did not exceed 2 standard deviations of the mean weight, and the mean body weights for each group of each sex were not statisticallydifferentat the 5% probabilitylevel. Page 10 of 38 HWI 6329-159 Study Design Animals weighing from 2,523 to 3,276 g at initiationof treatmentwere placed into the following study groups: Group Dose Test/Control Dose Level Volume Number of Animals' Material (mg/kq) (mL/kq) Males Females 1 (Control) Sterilewater 0 0.5 4 4 2 (Low) T-6246 5 0.5 4 4 3 (Medium) T-6246 10 0.5 4 4 4 (High) T-6246 100 0.5 4* 4 5 (High) T-6246 Soo 0.75 4* 4 one animal sacrificedafter treatment due to a possible broken back and replaced with another male animal. a Two animals/sex/doselevel were sacrificedon Day 15. The remaining animals (two animals/sex/doselevel)were sacrificed on Day 28. Justificationfor SpeciesSelection Historically,the New ZealandWhite albino rabbit has been the animal of choice because of the large amount of backgroundinformationon this species. PROCEDURES Dose Preparation and Administration The test materialwas dilutedwith sterilewater to achievea specific concentrationfor each dose level in Groups 2 to 5. An individualdose of each respectivetest solutionor controlwas calculatedfor each animal based on its body weight on the day of treatment. The respectivetest solutionwas administeredby intravenousinjectioninto the marginalear vein of the right ear over approximately22 to 60 seconds. The preparedtest solutionswere stored at room temperatureuntil administered. After administration,any remaining test solutionswere discarded. Reason for Route of Administration Intravenousinjection is an acceptableroute to assess systemicexposure. ooooIL7 Page 11 of 38 HWI 6329-159 Observations of Animals Clinical observationswere conductedpredose and at approximately0.5, 2, and 4 hours after intravenousinjection. Additionalclinicalobservationsand twice a day mortality checkswere cotiductedaily thereafteruntil the scheduledsacrificeinterval(Day 15 or Day 28). Body weights were determined on Day -7 for randomization purposes and before test or controlmaterialadministration(Day 1). Additionalbody weightswere determinedat the scheduledsacrificeinterval(Day 15 or Day 28) or at unscheduled sacrifices (when survivalexceeded 1 day). Sami)leCollections A blood sample (approximately4 mL) was collected from the marginal ear vein (leftear) of the animalsat 4-, 8-, 12-, 24-, and 48-hourspost-injection, and on Day 8. An approximate4-mL blood samplewas also collectedon Days 15 and 22 for the animalsscheduledfor sacrificeon Day 28. In addition,at the time of necropsy, approximately20 mL of blood was obtainedfrom the posterior vena cava of each animal. All sampleswere stored at room temperature,and then centrifuged,separatedinto serum and cellular fractions. These samples were then stored in a freezer set to maintain a temperatureof -200C 100C until shipped to the Sponsor. Pathology The animals sacrificedon Days I and 2 due to apparent broken backs were necropsied in the same manner as the animals surviving to the scheduled sacrificeswith the exceptionthat they received an abbreviatedgross necropsy examination and any abnormalitieswere recorded. On Day 15, the first two animals/sexassigned to each dose level (basedon the group assignment randomization)were anesthetizedwith sodium pentobarbital (via injectionin the marginalear vein), bled via the posteriorvena cava, and exsanguinated. An abbreviatedgross necropsy examination was not done, however,tissues were collected. The whole liver, bile, and both kidneys from each animal were collected,weighed (volumeonly determinedfor bile), and immediately placed in a freezer set to maintain a temperatureof -200C IOOC. After tissue/bilecollection,the animalswere discarded. The remainingtwo animals/sex/doselevel were anesthetized,bled, and exsanguinatedon Day 28 in the same manner as the animals sacrificed on Day 15. ooools Page 12 of 38 HWI 6329-159 ShiRment of Tissues After completion-ofthe in-lifephase, the blood samples (serumand cellular fractions),livers,bile,and kidneyswere sent frozen (on dry ice) to the Sponsor (James D. Johnson, 3M E.E. & P.C., Bldg. 2-3E-09, 935 Bush Avenue, St. Paul, MN, 55106), along with their correspondingweights or volumes. The Sponsor is responsiblefor the retentionand dispositionof the samples-. HWI does not accept any responsibilityfor the analysis of the samples collectedin this studynor are theseresultspresentedin this report. StatisticalAnalyses No statisticalanalyseswere requiredby the protocol. Location of Raw Data, Records, and Final ReRort The raw data, records,and an originalsigned copy of the final reportwill be retained in the archives of HWI in accordancewith HWI SOP. Page 13 of 38 HWI 6329-159 RESULTS Body Weights Individualbody weights are in Table 1. There was no meaningfuleffect on body weight gain during the study. Clinical Observations Individualclinicalsigns are in Table 2. All animalsappearednormal throughoutthe study with the followingexceptions: One Group 4 male (No. F54106)treated with T-6246 at 100 mg/kg appeared to have injured its back on Day 2 and was sacrificed,necropsied,and replaced with No. F54099. The replacement animal appeared normal throughoutthe study. One Group 5 male (No. F54076) treated with T-6246 at 500 mg/kg appeared to have injured its back on the day of treatmentand was sacrificed, necropsied, and replaced with No. F54112. The replacement animal appeared normal throughout the study. Pathology Individualanimal pathology commentsare presented in Table 3. Individual animal tissue weights and bile volumesare in Table 4. The necropsyof animal No. F54106 revealed the skeletalmuscle surroundingthe lumbar spinal cord was diffuselydark red. In animal No. F54076,the skeletalmuscle surrounding both hind limbs and the sacral spinal cord was diffuselydark red. The remaining animals survivedto their scheduled sacrificeand were not examined grossly, although tissues were saved. Page 15 contains a pathologyreport by the study pathologist. DISCUSSION The level of systemic exposure of T-6246 was evaluated in male and female albino rabbitswhen administeredas a single intravenousinjectionat levels of 5, 10, 100, and 500 mg/kg. There were no test material-relatedeffects in any of the animalsfollowingadministrationof this material. 0()0()20 Steven M. Glaza Study Director Acute Toxicology Page 14 of 38 SIGNATURE Date HWI 6329-159 Page 15 of 38 HWI 6329-159 PATHOLOGY REPORT There were two male rabbits sacrificedon Day 1 or Day 2 and necropsied. At necropsy, the skeletalmuscle surroundingthe lumbar spinal cord in Animal No. F54106 (100 mg/kg of body weight) was diffuselydark red. In Animal No. F54076 (500 mg/kg of body weight), the skeletalmuscle surroundingboth hind limbs and the sacral spinalcord was diffuselydark red. The findings in these animals are indicativeof injury of undeterminedetiologyto the caudal region of the back. The liver, bile, and both kidneys from these animalswere collected as requiredby protocol. After necropsy,the animals were discarded. Thoma,-Z. Pwi-m-er-,pbo Date Pathologist (6329-159.lhm) 051295 0()00;!2 Page 16 of 38 HWI 6329-159 Table 1 IndividualBody Weights (g) Random- Animal ization Initial Terminal Sex Number (Day -7) (Day 1) Day 15 Day 28 Group 1 (Control)- SterileWater for Injection(0 mg/kq) Male Female F54105 F54075 F54081 F54107 F54104 F54086 F54074 F54085 2,603 2,531 2,373 2,544 2,665 2,526 2,540 2,553 2,761 2,539 2,526 2,731 2,890 2,632 2,703 2,667 2,933 2,703 - 3,130 2,775 - 2,874 3,056 2,990 3,014 Male Female F54083 F54100 F54077 F54094 F54091 F54108 F54102 F54097 Group 2 2,381 2,675 2,459 2,466 2,717 2,412 2,690 2,439 T-6246 (5 mg/kq) 2,598 2,869 2,592 2,601 2,538 3,030 - - 2,785 2,622 2,839 2,533 2,996 2,790 - - 2,859 2,916 3,290 2,928 Male Female F54071 F54093 F54095 F54101 F54110 F54096 F54090 F54078 GrouR 3 T-6246 (10 mg/kg) 2,487 2,652 2,662 2,622 2,523 2,776 2,772 2,642 2,709 2,993 - - 2,719 2,772 2,542 2,710 2,802 2,914 2,740 2,842 2,959 3,022 - 3,126 3,162 3,022 3,244 - Not required. ()000213 Sex Male Female Page 17 of 38 HWI 6329-159 Table 1 (Continued) IndividualBody Weights (g) Animal Number Randomization (Day -7) Initial Terminal (Day 1) Day 15 Day Group 4 - T-6246 (100-mg/kq) F54088 F54106a F54089 F54087 F54099 2,494 .2,677 2,597 2,578 2,360 2,582 2,833 2,662 2,665 2,764 2 771 2:803(') 3,004 - 2,911 3,060 - F54080 F54092 F54109 F54114 2,498 2,400 2,592 2,776 2,622 2,540 2,657 2,933 2,835 2,667 - - 2,979 3,296 Male Female Group 5 T-6246 (500 mg/kq) F54076' F54113 F54082 F54111 F54112 2,655 2,667 2,734 2,547 2,996 2,678 2,753 2,843 2,784 3,276 - 2,896 - 3,530 F54098 F54084 F54079 F54116 2,565 2,510 2,468 2,442 2,679 2,600 2,526 2,611 2,856 2,821 - 3,216 3,228 - 2,959 3,002 - Not required. a Animal No. F54106 was originally selected by the randomization program for use in the study and was treated. This animal was sacrificedon Day 2 due to a broken back and was replaced with No. F54099. Number in superscriptedparentheses indicates the day the body weight was taken. b Animal No. F54076 was originally selected by the randomization program for use in the study and was treated. This animal was sacrificed on Day I due to a broken back and was replacedwith No. F54112. oooo%4 Sex Male Female Male Female Page 18 of 38 HWI 6329-159 Table 2 IndividualClinicalSigns Animal Number Observation Hour (Day 1) 0.5 2 4 Day 2 3 to 15 16 to 28 Group 1 (Control)- SterileWater for Injection(0 mg/kq) F54105 Appeared normal F54075 Appeared normal F54081 Appeared normal F54107 Appeared normal F54104 Appeared normal F54086 Appeared normal F54074 Appeared normal .1 F54085 Appeared normal Group 2 - T-6246 (5 mcl/kq) F54083 Appeared normal ri .1 F54100 Appeared normal t .1 F54077 Appeared normal F54094 Appeared normal F54091 Appeared normal F54108 Appeared normal vi F54102 Appeared normal F54097 Appeared normal Animal sacrificedon Day 15. Condition existed. 0()00%5 Page 19 of 38 Sex Male Female Male Female Table 2 (Continued) IndividualClinicalSigns. Animal Number Observation Hour (Day 1) 0.5 _L 4 2 Group 3 - T-6246 (10 mg/kq) F54071 Appeared normal F54093 Appeared normal F54095 Appeared normal F54101 Appeared normal F54110 Appeared normal I/ F54096 Appeared normal F54090 Appeared normal 8( F54078 Appeared normal Group 4 - T-6246 (100 mg/kq) F54088 Appeared normal F54106 Appeared normal .1 Broken back - Moribund sacrifice F54089 Appeared normal F54087 Appeared normal F54099a Appeared normal vi F54080 Appeared normal .1 F54092 Appeared normal F54109 Appeared normal F54114 Appeared normal 1( HWI 6329-159 Day 3 to 15 16 to 28 vi Animal sacrificedon Day 15. Condition existed. Conditionnot evident. a Replacement animal for Animal No. F54106. o 0%;: Sex Male Female Page 20 of 38 Table 2 (Continued) IndividualClinical Signs Animal Number Observation Hour (Day 1) 0.5 2 ' 4 2 Group 5 - T-6246 (500 mg/kq) F54076 Appeared normal Broken back t Moribund sacrifice F541 13 Appeared normal F54082 Appeared normal F54111 F54112 a Appeared normal Appeared normal F54098 Appeared normal F54084 Appeared normal F54079 Appeared normal F54116 Appeared normal HWI 6329-159 Day 3 to 15 16 to 28 Animal sacrificed on Day 15. Condition existed. Condition not evident. t Condition evident at time of 12-hour bleeding interval. a Replacement animal for Animal No. F54076. 10()o()z7 Page 21 of 38 Table 3 Individual Pathology Comments HWI 6329-159 Animal Test Day Number Sex Died Sacrificed Necropsy Observation Group 4 T-6246 (100 mg/kq) F54106 m 2 The skeletal muscle surroundingthe lumbar spinalcord is diffusely dark red. F54076 m Group 5 1 T-6246 (500 mg/kq) The skeletal muscle surrounding both hind limbs and sacral spinal cord is diffuselydark red. oooOZ8 Page 22 of 38 HWI 6329-159 Table 4 Individual Animal Tissue Weights and Bile Volumes Animal Sacrifice Weight (g) Bile -Sex Number Day Liver Kidneys Volume (mLl Group I (Control)- SterileWater for Injection(0 mg/kq) Male F54105 15 86.35 14.92 0.6 F54075 15 60.25 15.97 0.1 F54081 28 80.597 14.764 1.2 F54107 28 80.649 15.620 0.4 Female F54104 15 98.40 16.03 0.7 F54086 15 77.30 15.23 0.7 F54074 28 82.895 18.793 1.6 F54085 28 80.950 14.925 1.3 Group 2 T-6246 (5 mgZXgl Male F54083 15 73.15 14.87 0.7 F54100 15 77.90 17.62 0.7 F54077 28 75.699 17.864 0.3 F54094 28 78.689 17.213 0.8 Female F54091 15 84.90 14.92 1.6 F54108 15 79.90 15.40 0.4 F54102 28 101.494 18.308 1.7 F54097 28 73.621 16.214 1.4 oo002!i -Sex Male Female Page 23 of 38 HWI 6329-159 Table 4 (Continued) IndividualAnimal Tissue Weights and Bile Volumes Animal Number Sacrifice Day Weight (g) Liver Kidneys Bile Volume (mL) Group 3 - T-6246 (10 mg/kq) F54071 15 77.10 15.88 0.5 F54093 is 81.40 15.50 0.8 F54095 28 85.504 15.561 1.2 F54101 28 85.668 16.120 1.0 F54110 15 83@68 15.79 1.4 F54096 15 78.10 18.70 1.1 F54090 28 76.220 15.878 2.1 F54078 28 83.460 18.130 1.0 Group 4 T-6246 (100 mg/kq) Male F54088 15 84.26 16.98 0.4 F54106 2 82.301 16.550 2.0 F54089 28 71.796 16.434 1.0 F54087 28 83.710 18.591 0.6 F540 gga 15 82.143 16.038 0.5 Female F54080 15 75.42 18.32 1.2 F54092 15 78.36 16.68 F54109 28 73.958 16.915 1.6 F54114 28 80.966 19.404 2.0 a Replacement animal for Animal No. F54106. This animal had no gallbladder,so no bile was collected. -Sex Male Female Page 24 of 38 HWI 6329-159 Table 4 (Continued) IndividualAnimal Tissue Weights aiiuu,ic volumes Animal Number Sacrifice Day Weight (g) Liver Kidney Bile Volume (mL) Group 5 - T-6246 (500 mg/kq) F54076 1 78.058 14.695 2.0 F54113 15 91.52 16.57 1.0 F54082 28 88.965 19.431 1.6 F54111 28 83.823 19.405 1.5 F54112a 15 97.824 21.069 1.4 F54098 15 78.95 14.72 1.2 F54084 15 66.42 13.18 0.8 F54079 28 83.117 14.785 1.8 F54116 28 71.962 15.793 2.0 a Replacement animal for Animal No. F54076. 0 0 IL Page 25 of 38 APPENDIX A Protocol Deviations Protocol TP8084.PK Protocol Amendment No. 1 HWI 6329-159 oooo.,32 Page 26 of 38 Protocol Deviations HWI 6329-159 Protocol Page 6, 7. ExperimentalDesign, C. Dosing Procedures,(1) Dosing Route. Intravenous injectioninto the marginalear vein of the right ear over approximately30 to 60 seconds. Actual Procedure - The rate of injectionfor one Group 5 female (No. F54098) was 22 seconds. Also, the time for dose administration for one male animal (Group4, No. F54087) was incorrectly recorded thus the exact duration of dosing can not be detemi ned. These deviations are not consideredto have had an adverse effect on the outcome of the study. CoOO33 HAZLUNCCN W W IS C 0 N S IN POST OFFICR BOX 75,IF) MADISOtl,Wl FP370,' 15-15 Page 27 of 38 a CORNING Cofnpany Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.EXT Study Title: Single-Dose Intravenous Phamacokinetic Study of T-6246 in Rabbits Date: March 30, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-159 0()0034- P iio n e 6 0 8 .2 4 1 .14 1 E X P R E S S tv!A 1I- D I I I F R y @iI K IN S tvlA N BLVD F a x 608 2 4. 1. -.7 2.2 M A D I S C)N. w 1 53 704 Page 28 of 38 STUDY IDENTIFICATION TP8084.EXT Page 2 Single-Dose Intravenous Pharmacokinetic Study of T-6246 in Rabbits HWI No. Test Material Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date 6i29-159 T-6246 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, WI 53704 April 4, 1995 May 1, 1995 June 12, 1995 e,o. 0 3 5 Page 29 of 38 TP8084.EXT Page 3 1. Studv Single-Dose IntravenousPharmacokineticStudy in Rabbits 2. Purpose To assess the level of systemicexposurewhen the test material is administeredas a singleintravenousinjectionto rabbits 3. Regulatory Compliance This study will be conductedin accordancewith the followingGood LaboratoryPracticeRegulations/Standards/Guidelinweisth the exceptionthat analysisof the test materialmixturesfor concentration,solubility,homogeneity,and stabilitywill not be conducted: ( ] Conduct as a NonregulatedStudy CXI 21 CFR 58 (FDA) 40 CFR 160 (EPA-FIFRA) 40 CFR 792 (EPA-TSCA) C(81)30(Final)(OECD) 59 Nohsan No. 3850 (Japanese MAFF) NotificationNo. 313 (JapaneseMOHW) All proceduresin this protocol are in compliancewith the Animal WelfareAct Regulations. In the opinionof the Sponsorand study director,the study does not unnecessarilyduplicateany previous work. 4. Ouality Assurance The protocol,studyconduct,and the final reportwill be auditedby the Quality AssuranceUnit in accordancewith Hazleton Wisconsin (HWI) StandardOperatingProcedures(SOPS) and policies. 5. Test Material A. Identification T-6246 B. PhysicalDescription (To be documentedin the raw data) C. Purityand Stability The Sponsorassumesresponsibilityfor purityand stability determinations(including.undetrest conditions).Samplesof test material/vehiclmeixture(s)for concentrations,olubility, homogeneity,and stabilityanalyseswill be taken before administrationif requestedby the Sponsor. These samples(if taken)will be sent to the Sponsorafter experimental termination. D. Storage Room temperature ej(,)OO36 Page 30 of 38 TP8084-EXT Page 4 E. Reserve Samples Reservesample(s)of each batch/lotof test and control materialswill be taken for this study. The test and controlmaterialreservesampleswill be stored at HWI in a freezerset to maintaina temperatureof -20*C 10'C for 10 years per HWI SOP. The,Sponsorwill be contactedafter 10 years for dispositionin accordancewith the appropriate regulatoryGood LaboratoryPractices. F. Retention Any unusedtest materialwill be returnedto the Sponsorafter completionof the in-lifephaseof the study. G. Safety Precautions As required by HWI SOPs and policies 6. ControlMaterial A. Identification Sterilewaterfor injection B. PhysicalDescription Clear,colorlessliqui'd C. Purityand Stability The purityand stabilityof this USP grade materialis consideredadequatefor the purposesof this study. D. Storage Refrigerated E. Reserve Samples See Section 5. E. Reserve Samples F. Retention Any remainingcontrolmaterialmay be used for other testing and will not be discardedafter issuanceof the finalreport. G. Safety Precautions As required by HWI SOPs and policies 7. ExperimentalDesign A. Animals (1) Species Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP,Inc. 0()0037 Page 31 of 38 TP8084.EXT Page 5 (3) Age at Initiation Adult (4) Weight at Initiation 2.5 to 3.5 kg (5) Number and Sex 20 males and 20 females (6) Identification Individualnumbered ear tag (7) Husband (a) Housing Individually,in screen-bottomstainlesssteel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds, Inc.). The food is routinelyanalyzedby the manufacturer for nutritionalcomponents and environmental contaminants. (c) Water Ad libitum from an automatic system. Samples of the water are analyzedby HWI for total dissolvedsolids, specifiedmicrobiologicalcontent, selectedelements, heavy metals, organophosphates, and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants in the food or water that would interferewith this study. (e) Environment Environmentalcontrols for the animal room will be set to maintain a temperatureof 19*C to 23*C, a relative humidity of 50% +2ffo,and a 12-hour light/12-hourdark cycle. (f)Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to HWI SOPS. An adequate number of extra animals will be purchased so that no animal in obviouslypoor health is placed on test. The animalswill be placed into study groups using a stratifiedbody weight randomizationprogram within nine days of study initiation. 0()003FJ Page 32 of 38 TP8084.EXT Page 6 (9) Justificationfor Species Selection Historically,the New Zealand White albino rabbit has been the animal of choice because of the large amount of background informationon this species. B. Dose Administration (1) Test Groups Grout) Test/Control Material- Dose Level (mg/kq)' I (Control) Sterilewater 0 2 (Low) T-6246 5 3 (Medium) T-6246 10 4 (High) T-6246 100 5 (High) T-6246 500 Number of Animals Mal es Females 4 4 4 4 4 4 4 4 4 4 a The dose volume will be 0.5 mL/kg of body weight. b Two animals/sex/doselevel will be sacrificedon Day 15. The remaining animals (two animals/sex/doselevel) will be sacrificed on Day 28. C. Dosing Procedures (1) Dosing Route Intravenousinjectioninto the marginal ear vein of the right ear over approximately30 to 60 seconds. (2) Reason for Dosing Route Intravenousinjectionis an acceptableroute to assess systemic exposure. (3) Dosing Duration Single dose (4) Dose Preparation The day of treatmentwill be designated as Day 1. The Group I animals will be treated with sterilewater at a dose volume of 0.5 mL/kg. The test materialwill be diluted with sterile water to achieve a specific concentrationfor each dose level in Groups 2-5. Individualdoses will be calculatedbased on the animal's body weight taken just before test material administration. The prepared test mixtureswill be stored at room temperatureuntil administration. Page 33 of 38 D. Observation of Animals TP8084.EXT Page 7 (1) Clinical Observations The animalswill be observed for clinical signs of toxicitybefore test or control material administration, at approximately0.5, 2.0, 4.0 hours post-injection (Day 1), and daily thereafterfor clinicalsigns and twice daily (a.m.and p.m.) for mortalityuntil the scheduled sacrifice interval(Day 15 or Day 28). Observationsmay be extended when directed by the Study Director. (2) Body Weights For randomization,before test or control material injection(Day 1), at the scheduledsacrificeinterval (Day 15 or Day 28), and at unscheduled death and sacrifices (when survivalexceeds 1 day) (3) Sami)leCollections (a) Frequency 4-, 8-, 12-, 24-, and 48-hours post-injection,on Days 8, 15, 22, and at the scheduled sacrifice interval (Day 15 or Day 28) (b) Method of Collection/Number of Animals Blood samples (approximately4 mL) will be collected from the marginalear vein (leftear) of all animals at 4-, 8-, 12-, 24-, and 48-hourspost-injection,and on Day B. Additional samples will be collectedon Days 15 and 22 for the animals scheduled for sacrifice on Day 28. Approximately20 mL of blood (actual volume to be documented in the raw data) will be obtained from the posterior vena cava at the time of the scheduled sacrifice (Day 15 or Day 28). Approximately20 mL of blood will be collected from moribund animals during the study, also, if possible. The samples will be stored at room temperature and then centrifuged,and the separate serum and cellular fractionsstored in a freezer set to maintaina temperature of -20*C 10*C. The separated serum and cellularfractionswill be sent frozenon dry ice to the Sponsor after experimental termination. The Sponsor is responsiblefor the retentionand dispositionof the samples. Page 34 of 38 Samples will be shipped to: TP8084.EXT Page 8 James D. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN .55106 James D. Johnson or his alternatewill be notified by telephoneat (612)778-5294 prior to the shipment of the samples. E. Termination (1) Unscheduled Sacrifices and Deaths Any animal dying during the study or sacrificedin a moribund conditionwill be subjectedto an abbreviated gross necropsy examinationand all abnormalitieswill be recorded. Animals in a moribund conditionwill be anesthetizedwith sodium pentobarbital(via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. Tissues, as described in section 7.E. (3) Sample Collection,will be collectedfrom any animal dying during the study or sacrificed in a moribund condition. After necropsy,the animalswill be discarded. (2) Scheduled acrifices On Day 15, the first two animals/sexassigned to each dose level (based on the group assignmentrandomization)will be anesthetizedwith sodium pentobarbital(via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. The remaining two animals/sex/doselevel will be anesthetizedwith sodium pentobarbital(via injectionin the marginalear vein), bled via the vena cava, and exsanguinatedon Day 28. An abbreviated gross necropsy examinationwill not be done, however, tissues (as describedin section7.E. (3) SampleCollection)will be collected. (3) Sample Collection The whole liver, bile, and both kidneys from each animal will be collected,weighed (volumeonly determined for bile), and immediatelyplaced in a freezer set to maintain a temperatureof -20*C 10*C. After sample collection, the animals will be discarded. The samples (liver,bile, and kidneys)will be sent frozen on dry ice to the Sponsor after experimentaltermination. The samples and their corresponding weights or volumes will be shippedto the person listed in Section 7.D.(3).(b). The Sponsor is responsiblefor the retention and disposition of the samples. ---------- Page 35 of 38 F. StatisticalAnalyses No statisticalanalyses are required. TP8084-EXT Page 9 8. Report A final report includingthose items listed below will be submitted. Descriptionof the test and control materials Descriptionof the test system Procedures Dates of experimentalinitiationand termination Descriptionof any toxic effects Gross pathologyfindings (if applicable) Gross pathology report (if applicable and requested by the Study Director) Individualanimal tissue weights and bile vol.umes 9. Location of Raw Data, Records.-and Final Report Originaldata, or copies thereof,will be availableat HWI to facilitateauditing the study during its progressand before acceptanceof the final report. When the final report is completed, all originalpaper data, includingthose item listed below will be retained in the archives of HWI according to HWI SOP. Protocol and protocol amendments Dose preparation records In-liferecords Body weights Dose administration Observations Sample collection records Shipping records Pathology Records Study correspondence Final report (originalsignedcopy) The followingsupportingrecordswill be retainedat HWI but will not be archivedwith the study data. Animal receipt/acclimationrecords Water analysis records Animal room temperature and humidity records Refrigerator and freezer temperature records Instrument calibration and maintenance records Page 36 of 38 PROTOCOL APPROVAL TP8084.EXT Page 10 John L. Butenhoff,PhD Sponsor's Representative 3M %\ - Tteven M. Glaza ci Study Director Acute Toxicology Hazleton Wisconsin, Inc. - @wc., A46-e,@ Representatpve Quality Assurance Unit Hazleton Wisconsin,Inc. (6329-159.protdsk2) Date -5--1ri- is Date 3 -3c) Date oo0043 Page 37 of 38 HAZLEEC(ON W W IS C 0 N S IN POST OFFICE BOX 7545 MADISON,WL 53707-7545. a CORNING Company I PROTOCOL TP8084.EXT Single-Dose Intravenous PharmacokineticStudy of T-6246 in Rabbits HWI 6329-159 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E.-02 P.O. Box 33220 St. Paul, MN 55133-3220 Sponsor's Representative John L. Butenhoff, PhD Contractor Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wl 53704 Study Director Steven M. 61aza Amendment No. I This amendment modifies the following portions of the protocol: EffectiveApril 4, 1995 1. Page 6. 7. ExperimentalDesign; B. Dose Administration; (1) Test Groups. The test material mixture for Group 5 could not be prepared at the concentration needed to utilize a dose volume of 0.5 mL/kg. Modify footnote "a" in this sectionwith the followingunderlinedaddition: a The dose volume will be 0.5 mL/kg of body weight for Groups 1-4 and 0.75 mL/kq for Group 5. P h o n e 6 0 8 -2 4 1 -14 7 1 E x P R E S S M A IL D E L !V E R Y 330 1 -KINSMAN BLVD Fa x MADISON. 6 0 8 -2 4 1 -7 2 2 7 Wi 53 704 0()004,,l Page 38 of 38 Amendment No. 1 HWI 6329-159 Page 2 EffectiveApril 5, 1995 2. Page 6. 7. ExperimentalDesign; B. Dose Administration;(1) Test Groups. Animal No. F54076 (Group 5 male) and Animal No. F54106 (Group 4 male) were sacrificedon Days I and 2, respectively,due to injuries (apparent broken backs). Add the followingparagraph to this section: Due to the sacrificeon Day I of one Group 5 male (Animal No. F54076) and on Day 2 of one Group 4 male (Animal No. F54106) because of injuries (apparent broken backs), replacementanimals will be treated at the same dose levels in the same manner as for the initial animals in the study. The observations(clinicalobservations,body weights and sampl6 collections)and the terminationof the animals (unscheduled sacrificesand deaths, scheduled sacrifices, and sample collection)will be conducted in the same manner as for the other animals in the study terminating on Day 15. PROTOCOL AMENDMENT APPROVAL 2- John L. Butenhoff, PhD Date Sponsor's Representative 3M Toxicology Service Medical Department Steven M. Glaza u Study Director Acute Toxicology Hazleton Wisconsin, Inc. -@t4 Reprei-elitatiAe Quality AssUrance Unit Hazleton Wisconsin, Inc. (6329-159.Aml.dsk3) Date -7Date oooo@Ll%- I 9.1.2 AmalyticalprotocolAMDT-042095.1 ()()0041 3M Environmental Laboratory Protocol- AnalyticaSltudy Single-dosIentravenousPharmacokineticStudy ofT-6246inRabbits In-VivoStudy ReferenceNumber: HWI#6329-159 Study Number: AMDT-042095.1 TestSubstance:FC-95 (T-6246) Name and Address ofSponsor: 3M SCD Division 367 Grove Street St.Paul,NIN 55106 Name and Address of TestingFacility: 3M EnvironmentalTechnologyand Services 935 Bush Avenue St.Paul,NfN 55106 Proposed InitiatioDnate: July25, 1995 Proposed Completion Date: August 25, 1995 Method Numbers AMDT-M-1-0, AMDT-M-2-0, AMDT-M4-0, AMDT-M-8-0, AMDT-M-14-0, and Revisions: Thermal ExtractionofFluorideby Means ofa Modi ca Dohrmann DX2000 OrganicHalideAnalyzer-Liver FluorideMeasurement by Means of an OrionEA940 Expandable Ion Analyzer Extractionof Fluorochemicalsfrom RabbitLiver AnalysisofFluorideUsing theSkalarSegmented Flow Analyzer with Ion SelectivEelectrode Thermal ExtractionofFluorideby Means ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer-Serum Author:James D. Johnson Approved ames ]@.@hnson @@sylitudMyector 1. Date John Butenhoff,PhD Date Sponsor 000047 1.0PURPOSE Thisstudyisdesignetdoprovidienformatiaosntowhethetrheperfluorooctanesulfonataenion does go totherabbitliverand othertissueswhen the material isadministeredinan intravenousdose,and to ascertainthechange inconcentration with time afterdose inserum and liver. 2.0 TEST MATERIALS 2.1Test,Control,and Reference Substancesand Matrices 2.1.1AnalyticalReference Substance: FC-95, lot161 or 171.They are equivalent. 2.1.2AnalyticalReference Matrix: Bovine liverand bovine serum 2.13 AnalyticalControl Substance: None 2.1.4AnalyticalControl Matrix: Bovine liverand bovine serum 2.2 Source of Materials:3M ICP/PCP Division(2.1.1),grocerystore(2.1.2,2.1.4 liver)S,igma Chemical Company (2.1.22,.1.4serum) 2.3Number of Test and Control Samples: Liverand serum from 32 testanimals and 8 controlanimals.Two animalswere replacedat24 hours(animalF54076 was replacedwith animalF54112, and animalF54106 was replacedwith animal F54099). Otherbiologicatlissue(skidney,bile,cellulafrractionw)illbe available foranalysisifdeemed appropriatbey the Study Director. 2.4 Identificatioonf Test and Control Samples: The samplesareidentifieudsing theHWI animalidentificatinounmber which consistsofa lettearnd fivedigit number,plusthetissueidentitaynd day identit(yserum). 2.5Purityand StrengthofReference Substance:To be determinedby Sponsor. 2.6StabilityofReference Substance: To be determinedby Sponsor. 2.7StorageConditionsforTest Materials:Room temperature(2.1.1), -20 IOOC (2.1.22,.1.4).Testand Controlsampleswillbe receivedaccordingto AMDT-S- 10-0. ,()OoklS 2 2.8DispositioofnSpecimensB:iologictailssuaensdfluidwsillberetainpeedr GLP Regulationforthetime periodrequiredforstudieslongerthan 28 days. 2.9SafetyPrecautions:RefertoappropriatMeSDS. Wear appropriatleaboratory attireU.se cautionwhen handlingknivesforcuttingthe samples. 3.0 EXPERIMENTAL -Overview The tissuesfrom animalsdosed asdescribed(HWI#6329-159), areavailablfeor analysisforfluorinecompounds. At thediscretioonf theStudy Directora, seriesof analyticatlestscan be performed.The screeningforfluorideinliverviacombustion (SeeMethods--nextSection)istheappropriataenalysistopresentdefmitivedatafor fluorineintheliver.For theseanalysesb,ovine liverappearstobe a good approximationtorabbitliverformatrixspik@sand recoverydeterminationsS.ince perfluorooctanesulfonatneionisnotbiotransformedf,luorinecontentwillbe an accurateestimateof theconcentratioonf the compound. 4.0 EX]PERI[MENTAL -Methods 4.1Liver and Serum screeningmethods: (attached) 4.1.1AAMT-M-1-0, Thermal ExtractioonfFluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 4.1.2AAMT-M-2-0, FluorideMeasurement by Means ofan Orion EA940 ExpandableIon Analyzer 4.1.3AMDT-M-4-0, Extractioonf Fluorochemicalsfrom RabbitLiver 4.1.4AMDT-M-8-0, Analysisof FluorideUsing theSkalarSegmented Flow Analyzerwith Ion SelectivEelectrode 4.1.5AMDT-M-14-0, Thermal ExtractionofFluorideby Means ofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer-Serum 5.0DATA ANALYSIS 5.1Data Reporting: Datawillbe reportedasa concentratio(nweight/weighto)f fluoridpeer tissueor fluido,r as FC-95 pertissueor fluid.Statistiucsed,atthe discretioonfthe Study Directorm,ay includeregressioannalysisof serum concentrationwsith timeand averagesand standarddeviationosf concentrationfsor jot)00,1!) 3 differendtose groups.Ifnecessarys,implestatistictaelstssuch as Student'sttest may be appliedto determinestatisticdailfference.Half-lifoef disappearancefrom serum willbe determinedfrom thelinearregressionof theaveragesof the concentrationsatdifferentime pointsforthepostdistributiopnartof theserum concentrationversustime curve. 6.0 MAINTENANCE OF RAW DATA AND RECORDS 6.1 Raw Data and Records: Raw data,approved protocol,appropriatespecimens, approved finalreport,and electronidcatawillbe maintainedin theAMDT Archives. 7.0 REFERENCES 7.1 AMDT-S-10-0, Sample Tracking System 8.0 ATTACHMENTS 8.1AMDT-M-1-0, Thermal Extractionof Fluorideby Means of a Modified Dohrrnann DX2000 OrganicHalideAnalyzer-Liver 8.2 AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 8.3 AMDT-M4-0, ExtractionofFluorochemicalsfrom RabbitLiver 8.4AAMT-M-8-0, Analysisof FluorideUsing theSkalarSegmented Flow Analyzer with Ion SelectiveElectrode 8.5 AMDT-M-14-0, Thermal ExtractionofFluorideby Means of a Modified Dohrinann DX2000 OrganicHalide Analyzer-S'erum 4 3M Environmental Laboratory Method Thermal ExtractionofFluorideby Means of a ModifiedDohrmann DX2000 OrganicHalideAnalyzer - Liver Method IdentificatiNounmber: AMDT-M-1 RevisionNumber: 0 AdoptionDate: RevisionDate: None Author- Rich Youngblom Approved by: roupLea7"/ roliU@P L( /0 /3 Date QualityAssurance Date Software: MS Word 5.la AffectedDocuments: AMDT-M-2 FluorideMeasurement by Means of an OrionEA940 ExpandableIonAnalyzer AMDT-EP-3 RoutineMaintenance ofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer 1.0 SCOPE, APPLTCABLE COMPOUNDS, AND MATRTCES 1.1Scope: Thismethod isfortheoperatioonfa DohrrnannDX2000 when itisusedtoextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2Applicable Compounds: Fluorochen-dcalosrotherfluorinatecdompounds. 1.3Matrices:Biologicatlissuepsa,rticularlliyver. 2.0 KEYWORDS 2.1Fluoridef,luorine,xtractiopny,rolysiiso,nizatioino,nselectiveelectrodeD,ohrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS 3.1Glassware and exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizedgases,propercompressedgashandlingpracticersequired. 3.4Solventbased samplesmay flashm,ay needtoallowthem todry down beforestartinrgun. 3.5Potentiabliohazardsduetothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLIES AND MATERTALS 4.1Compressed Oxygen, Hydrocarbonfree,regulatetdo30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3QuartzglasssampleboatwithTeflonTmtubing,Dohrmann 890-097 or equivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012 orequivalent. 4.5Orion940999TotalIonicStrengtAhdjustmentBuffer(TISAB II)orequivalent. 4.6Sample collectiovnialsH,DPE. 4.7Milli-Qrmwater 4.8Polystyrenepipettes. 4.9ActivatedCharcoal,E.Merck 2005 orequivalent. 4.10Hamilton Syringeorequivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQUIPMENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzer,modifiedforfluorideextraction. 5.2IBM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluoridextraction. 5.4Excel Spreadsheet,version5.0orgreater 6.0 INTERFERENCES 6.1 Sample sizeislimitetdoapproximately150mg, dependingon samplemoisturecontent.This may varyfrom matrixtomatrix. 2 0()0()51 7.0 SAMPLE HANDLING 7.1Samples arenottobe handledwithbarehands. Fluoridemay leachfrom theskintothe sample. Use forcepsor probetotransfetrissues. 7.2Samples ofliverarecutfromfrozenliverand placedina taredand labeledweigh boat.Use a cleanscalpeland cuttinbgoard.The cuttinbgoardand scalpeslhouldbe cleanedwithwater, methanol,or methanol-watesrolutioanftereachliveriscut. 8.0 CALTBRATION AND STANDARDIZATION 8.1PreparatioonfCalibratiSotnandards 8.1.T1hestandarrdesquirfeodreachprojewcitlnleedtobeappropriaftoerthaitndividuparloject. Refertoprotocolforthatproject. 8.1.2Typically50-500ppm FC-95 inmethanolstandardsareused. 8.1.3Forrabbitlivesrtudiesu,sebeefliverasthematrix.Cuta pieceoffrozenbeefliver(100150mg) and weigh itina labeledandtaredweigh boat. 8.2 Calibratio-nOverview The normalcalibratiiosnthefluoridceurve(AMDT-M-2). However,ifanoptionaslpikedliver curveisrequiredtheprocedurelistebdelow isused. 8.2.1A calibratiocnurvefortheDX2000 isgeneratebdy spikingsampleswithknown standards and combusting them usingthesame methods and matrixtypeasthesamplestobe tested. 8.2.2Typicallyt,hreereplicateosfeachstandardand fiveconcentratioonfsstandardswillbe spiked. 8.2.3Standardcurvewillbe plotteadsMass SpikedF (ug)on thex-axisand StandardMass Recovered F (ug)on they-axis.Generatea regressiocnurveand calculatteheequationfortheline and ther2 value. 8.2.4Mass SpikedF (ug)= (Amount spikedinmL) x (Conc.of standaridnppm) x (0.6004)* *FC-95 is60.04% F therefor0e.6004isthefactorusedtoconvertFC-95 toF 8.2.5StandardMass RecoveredF (ug)= (TISAB volumeinML) x (Orionreadinginppm) 8.3Calibratio-nProcedure 8.3.1StartUp 8.3.1.1Run 2 ormore CleanCycleswhen startinignstrumenetachday.More cleancyclesmay be usediftheprevioussamplescontainedhighconcentratioonfsfluoride. 8.3.2Blanks 8.3.2.1Preparesampleusingthesame methods and typeofmatrixasthetestsample. 8.3.2.2Forrabbitstudiesu,sebeefliverasthematrix.Prepareatleast3 samplesofbeefliver (100- 150 mg) forblanks. 8.3.2.3Put sample inDohrrnannboat.Combust eachsample asdescribedinsection9.0and analyzesample accordingtomethod AMDT-M-2 fortheionselectiveelectrodaenalysis. 3 0()0051%- ---------:--, 8.3.2.4For rabbitstudiest,he meter readingfora blank sample should be 0.03 ppm or lower beforeproceedingwith the calibrationB.um samples untilthislimitisreached,or untilinthe judgement of theoperatorthe readingisstablewith respecttohistoricarleadings(previous48 hours). 8.3.2.5Fornon-rabbitstudiest,heblankreadingshouldreacha predetemiinedionconcentration beforeproceedingwith thecalibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwith thesample toaid combustion. 8.3.3 Standard Curve 8.3.3.1Weigh out atleast15 matrixsamples (5standardswith3 replicateesach)in taredand labeledweigh boats. For rabbitstudiesw,eigh 100-150 mg beef liversamples. Record weightsin studydata. Storethe matrixsamples on dry iceoricepacks tokeep them frozenuntilused. 8.3-3.2 Place weighed beef liversample inDohrmann sample boat. 8.3.3.3Startwith theloweststandardconcentratiorlUsing a Hamilton syringe,ejecta fixed quantityof thestandardon or inthe matrix.For rabbitstudies,use 4 uL ofstandardand ejectiton or inthebeef liver. 8.3.3.4At least3 replicatesshouldbe used forthelowest standardconcentrationm;ore replicates may be used atthediscretionofthe analyst. 8.3.3.5Combust the sample asdescribedinsection9.3 and analyzeaccordingto AMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessignificantldyifferenftrom theothertwo replicatesn,m anothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe old replicateand thatthenew replicatweillbe used to calculatetheregressioncurve. 8.3.3.7Vvrhenallstandardshave been run,calculatethe r2. r2 must be atleast0.95. Ifitisnot at least0.95,consultwith supervisor. 8.3.3.8A new standardcurve shouldbe nm when the combustion tubeor sample matrix is changed. New standardcurve may alsobe run atthe discretioonf the analyst. 8.4 Storage Conditions for Standards 8.4.1Storage requirementsforstandardsare dependent on the individualstandardsused. Typically,standardsarestoredatroom temperatureinplasticscrew top bottles. 8.4.2 New FC-95 standardsshouldbe preparedatleastonce a month. 9.0 PROCEDURES 9.1Typical Operating Conditions: 9.1.1Combustion tube temperature= 950'C. 9.1.2Oxygen and Helium flow 50 cc/minute. 9.1.3Vaporization/Dryingtime 240 seconds. 9.1.4Bake time = 300 seconds. 9.2 Start Up Procedure: 9.2.1Ifthe program isnot starteds,tarttheEOX program on the PC. 9.2.2Open the SYSTEM SETUP window. 9.2.3Put the furnacemodule and the cellintheREADY mode. 9.2.4Close the SYSTEM SETUP window. 0 000 4 9.2.5When the oven has reachedthe READY temperature,run the CLEAN found in theCELL CHECK menu. 9.2.6 See AMDT-EP-3 fordetailosf theDohrmann software. BOAT program 9-3Sample ExtractionProcedure: 9.3.1Open theSAMPLE HATCH and placethesample in theBOAT. Itmay be necessaryto rrdxapproximately50 mg of charcoalwith thesample toaid combustion. Ifthisisdone,charcoal should alsobe mixed inwhile establishintghebaselineand when generatingthe standardcurve. 9.3.2 Close SAMPLE HATCH. 9.3.3 Add appropriatevolume ofTISAB solutionor 1:1 TISAB:Milli-QTI"water mixturetoa labeledsample collectiovnial.Typically0.6mL to 15 mL areused. For rabbitstudiesu,se 1.0or 2.0 mL of 1:1 TIS AB: Milli-QTI"water n-dxture. 9.3.4 Placethevialso thatthetipoftheCONMUSTION TUBE isintheTISAB atleast0.25 inches.Gases releasedduringpyrolysismust bubble through the TISAB. 9.3.5 Run the EOX-SOLIDS program found in theRUN menu. 9.3.6When the EOX program isfinishedr,emove the collectiovnialfrom the combustion tube. 9-3.7 IfundilutedTISAB was used tocollecthe sample,add an equalvolume of Nfilli-QTmwater to theTISAB tomake 1:1 TISAB:Nfilli-QTm. 9.3.8Rinse the end of the combustion tube with Milli-QTMwater and wipe with a KIMWIPE to remove any TISAB remaining on thetube. 9.3.9 Open the sample hatch and remove any remaining ash from the boat. Ash can be removed with a cottontippedapplicatoror vacuumed out. Itmay be necessarytoscrapparticleosffthe bottom with a spatulaor othersimilardevice.A drop of Milli-QTmwater may be added to the boat toaidinthe Clean Cycle. 9.3.10.Close the hatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample isreadyforanalysisby ionselectivelectrode(AMDT-M-2). 9.4 Sample Calculations 9.4.1 Use the standardcurve tocalculatethesample value. 9.4.2 Sample Mass Recovered F (ug)= (TISAB volinmL) x (Orionreadingin1212m-interc6po (Slope) 10.0 VALIDATION 10.1 QualityControl 10.1.1Daily StartUp Check Samples: Once the standardcurve isestablishede,ach day of analysisisstartedby analyzingQC samples. The QC samples are tobe the same as the lowest concentrationspiked samples used togeneratethestandardcurve. Each concentrationmust be done intriplicatuenlessthefirstwo replicateasrewithin20% of thestandardcurve,then a third replicatiesnot necessary. 10.2 Precisionand Accuracy: See method development analysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracy varieswhen analyzingsamples of different matricesand differenrteferencecompounds. 10.3 Other ValidationParameters: NA 5 0()00,k- 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1For thestandarcdurve,useregressioannalysiisnExcel,version5.0orgreater. 11.1.2To calculattehefluoridceontractioinnthesample,seemethod AMDT-M-2. 11.2 Analyzing theData 11.2.1r2must be atleast0.95orgreater".Outliersm"ay be excludediftwo ofthethreereplicates arewithin20% ofeachotherand theoutlieirsgreatetrhan200% oftheaverageofthosetwo or lessthan50% oftheaverageofthosetwo. Any suchoutliersshouldbe pointedoutinthedataand notedintheFinalReportalongwiththereasonitwas consideredan outlier. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1Rosemount Dohrmann D X2000 OrganicHalideAnalyzerOperator'Msanual (Manual 915349,revisioBn, December 1993) 13.2 AMDT-M-2 FluorideMeasurementby Means ofan OrionEA940 ExpandableIon Analyzer 13.3ANOT-EP-3 RoutineMaintenanceofa ModifiedDohnnann DX2000 OrganicHalide Analyzer 14.0 REVISIONS Revision Numbe Reason forChanp-e Revision Date 6 3M Environmental Laboratory Method FluorideMeasurement by Means ofan OrionEA940 ExpandableIon Analyzer Method IdentificatiNounmber: AMDT-M-2 RevisionNumber: 0 AdoptionDate: RevisionDate: None Author Rich Youngblom Approved By: G@, Leader 161-3 Date QualityAssurance Date Software: MS Word 5.1a AffectedDocuments: AMDT-M-1 Thermal ExtractioonfFluorideby Means ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer 1.0 SCOPE ,APPLICABLE COMPOUNDS, AND MATRICES 1.1SCOPE: Thismethod isforthecalibratiaond operationofan OrionEA940 Expandable Ion Analyzer. 1.2APPLICABLE COMPOUNDS: Fluoride. 1.3APPLICABLE MATRICES: LiquidsamplesinanappropriatbeuffersolutionP.referred pH of 6.0. 2.0 KEYWORDS 2.1Fluoridef,luorinei,onselectiveelectrode 3.0 PRECAUTIONS 3.1No hazardsidentifiweidththismethod. 4.0 SUPPLIES AND MATERIALS 4.1Orion940999TotalIonicStrengtAhdjustmentBufferH (TISABII)orequivalent. 4.2OrionModel 900001electrodfeillinsgolutio(nAgCl)orequivalent. 43 Orion940907 100 ppm fluoridsetandardorequivalent. 4.4Nfilh-QTMwateror equivalent. 4.5Magneticstirbars. 4.6Lab tissues. 4.7Sample collectiovnials. 4.8Plastic100mL volumetricflasks. 4.9Polystyrenepipettes. 4.10Miscellaneouslaboratorgylassware. 5.0 EQUIPMENT 5.1Orion Model EA940 ExpandableIon Analyzerorequivalent. 5.2Orion Model 960900 SolidStateCombinationFluoridelectrodoerequivalent. 5.3MagneticStirPlate. 5.4IBM compafible386 or486 computer(onlyneededifusingOrion3E software). 5.5OrionRS232 interfacceable(onlyneededifusingOrion3E software). 5.6MicrosoftExcel5.0(ordyneededifusingOrion3E software). 6.0 INTERFERENCES 6.1Itisrecommended thatthepH be atornear6.0.A 1:1 mixtureofTISAB and sample/MilliQT?,lwaterwillgenerallybringsampletopH of 6.0. 6.2Sample temperaturmeay effectfluoridmeeasurement. Itisrecormnendedthatthesamplebe atroom temperatureas thestandardswere when themeter was calibrated. 6.3The ratethesamplesarestirreadtshouldbe consistenwtiththeratethestandardswere stirred. 0()Oor 2 6.4 Air bubbles trappedunder electrodecan giveerroneousreadings.Make sureno airistrapped under electrode. 7.0 SAMPLE HANDLING 7.1 No specialhandlingnecessary. 8.0 CALTBRATION AND STANDARDIZATION 8.1 PreparationofCalibratioSntandards 8.1.1Measure50 mL ofTISAB ][iInto5 100niLplastivcolumetrifclasks. 8.1.L2abeltheflaskass0.05,0.1,0.5,1.0a,nd1.5ppm F-,alongwiththedateandyourinitials. 8.13Pipett0e.05,0.1,0.5,1.0a,nd 1.5mL of100ppm fluoridsetandaridntotheappropriately labeledflasks. 8.1.4Add approximately30 mL of Mlli-QTm watertoeach flask. 8.1.5Shake the flaskstomix the solutions. 8.1.6Eliminateairbubblesfrom the flasksby fippingthe flaskson theirsidesand rollingthe airin the flasksover theairbubbles. 8.1.7Bring the volume inthe flasksup tothe 100 mL mark with MiUi-QTM water. 8.1.8Invertand shake the flasksforthe finalmixing. 8.1.9Record standardsinStandardsLog Book. 8.2 Calibration 8.2.1Ifnecessary,remove tapefrom electrodefillinhgole. 8.2.2Invertprobe to wet top seal. 8.2.3Ejecta few dropsof fillinsgolutionfrom bottom of electrodeto wet lower seal. 8.2.4Filltheelectrodewith fillinsgolution. 8.2.5The meter and the F- electrodearetypicallcyalibratebdy directmeasurement with no blank correctionu,sing standardswith concentrationosf 0.05,0.1,0.5,1.0,and 1.5ppm F-,following themanufacturer'sinstructions.. 8.2.6Record the slopein theappropriatelog book. 8.2.7Clean theelectrodeby rinsingwith Nfilli-QTwMater and wiping the sidesdown with lab tissues. 8.3 Storage Conditions forStandards 83.1 Calibrationstandardsarestoredatroom temperature. 9.0 PROCEDURES 9.1 Calibrationand Measurement, Standard method: 9.1.1The sample tobe measured needs tobe n-Lixewdith TISAB using the proportions recommended by the TISAB manufacturer. 9.1.2Place a stirbar inthe sample and placethesample on thestirplate. 9.1.3Allow the sample torrdxfora few seconds beforeinsertingtheelectrode.When the electrodeisinsertedm,ake surethereareno airbubblestrappedunder theelectrode. 9.1.4The sample should be thesame temperatureasthe calibratiosntandardsand stirredatthe same rateas the calibratiosntandards. 9.1.5When the readinashave stabilizedr,ecordthereadingintheappropriatelog book. 3 0 0 0.'7!) 9.2CalibrationAnd Nleasurement,Using Orion 3E Software: 9.2.1Calibration: 9.2.1.1Follow steps8.2.1to8.2.4,. 9.2.1.2PressFunctionKey #8 (F8). 9.2.1.3The computer screenwillaskyou toconfirmthenumber of standardsto be used, concentratioonf thestandardsa,nd whetheror nota blankistobe includedinthecalibration. Make any necessarychangestotheinformationpresentedand clickon CONTINUE. 9.2.1.4Placetheelectrodeinthefirssttandardon thestirplateand clickon CONTINUE. 9.2.1.5Observe thereadingson thegraphicdisplayon thecomputer. When thereadingshave stabilizepdr,essACCEPT READING. 9.2.1.6Repeat step9.2.1.4and 9.2.1.5fortheremainingstandards. 9.2.1.7Afterthefinalstandardt,hecomputerwilldisplaytheslopeof thecurve,as wellas the intercepatnd correlationR.ecord theslope,intercepta,nd correlatioin theappropriatleog book and clickon CONTINUE. The calibratidoantaisautomaticallcyopiedtoC:\Orion\Data\Calib.txt. 9.2.2Data Spreadsheet: 9.2.2.1SelecteitherNEW orOPEN from theFILE menu toopen a new orexistingspreadsheet tostoredatain. 9.2.2.2Record thename ofthespreadsheetused intheappropriatleogbook. 9.2.3FluorideMeasurement: 9.2.3.1Follow steps9.2.1through9.2.4 9.2.3.2Enter thename ofthesample intheappropriatpelaceon thescreen. 9.2.3.3Clickon theNEW SAIviPLE button 9.2.3.4When thereadingshave stabilizecdl,ickon theRECORD buttonand writetheresultinthe appropriatleogbook. 10.0 VALIDATION 10.1 QualityControl: 10.2 Precisionand Accuracy 10.3 Other ValidationParameters Accordingto-Referenc1e3.2,therangeofdetectioins0.02 ppm fluorideup toa saturatesdolutionof fluoride. 11.0 DATA ANALYSIS 11.1 CalculationsNonenecessary. 11.2 AnalyzinFg--VtheData Nonenecessary. 12.0 ATTACHNIENTS None 13.0 REFERENCES 4 13.1Orion Model EA940 Expandable IonAnalyzerInstructioMnanual,Orion Research Incorporated1,991. 13.2Orion Model 960900 SolidStateCombinationFluorideElectrodeInstructioMnanual, Orion Research Incorporated1,991. 14.0 REVISTONS Revision Number Reason forChang-e Revision Date 0()00(,l 5 3M Environmental Laboratorl Method Extraction of Fluorochemicals from Rabbit Livers SOP IdentiricatioNnumber: AMDT-M-4 RevisionNumber: 0 Adoption Date: RevisionDate: None Author:Dave Christenson/CynthWieaber Approved By: /ruIp-eader /0 Date QualityAssurance Datf,- Software:MS Word, 6.0 AffectedDocuments: M-5, AnalysisofRabbitExtractforFluorochemicalsUsing Electrospray Mass Spectroscopy. oooo(@@-, 1.0 S OPE 1.1 Scope: Thismethod isfortheextractioonffluorochemicalfsrom rabbitlivers. Ethylacetateisused toextractfluorochemicalfsrom theliversforanalysisby electrospramyass spectroscopy. 1.2 ApplicableCompounds: Fluorochemicalsor otherfluorinatecdompounds. 1.3 Matrices:RabbitLivers. 2.0 KEYWORDS 2.1 Fluorochemicalsr,abbitliverse,lectrospramyass spectrometerf,luorinated compounds, extraction. 3.0 PRECAUTIONS 3.1 Use gloveswhen handlingtherabbitliverst,heymay containp.athogens. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1.1 Syringe,capableofmeasuring 100 p.L 4.1.2 Eppendorf typeor disposablepipets 4.1.3 Gloves 4.1.4 Plasticgrindingtubes 4.1.5 Plasticentrifugteubes,15 mL 4.1.6 Labels 4.1.7 Nitrogen 4.1.8 Timer 4.1.9 FiltersT,itannylonsyringefilter0s.,2gm. 4. 1.10 Analyticaplipetsg:lassvolumetdc pipets. 4.1.11 Disposableplasti3c cc syringes. 4.1.12 Crimp cap autovials. 4.2 Reagents 4.2.1 Aqueous Ammonium Acetate(Aldrich)a,pprox.250 ppm: Preparea 2500 ppm aqueous solutioonf ammonium acetateby adding250 mg ammonium acetatetoa 100 mL volumetdcflaskand dilutetovolume withMiUi-Q water.Dilutethissolution1:10 fora 250 ppm solution. 4.2.2 Sodium carbonate/SodiumBicarbonateBuffer(J.T.Baker), (Na@C03/NaHC03)0.25 M: Weigh 26.5g of sodium carbonate(Na@2C03) and 21.0g of sodium bicarbonate(NAHCO@ intoa 1 L volumetricflaskand bringtovolume withNEUI-Q water. 4.2.3 Diluteacetonitrisloelutiond,iluteacetonitri1l:e1withNOI-Q water. 4.2.4 EthylAcetate 4.2.5 Methanol 4.2.6 Milh-Q water 4.2.7 1H,lH,2H,2H -perfluorooctanesulfoanciicd(Aldrich) 4.2.8 FC-95 (3M SpecialtCyhemicalDivision) el()00(;13 2 5.0 EQUIPMENT 5.1 Ultra_Turra7x25 Grinderforgrindingliversamples. 5.2 Vortexmixer 5.3 Centrifuge 5.4 Shaker 5.5 AnalyticaElvaporator 6.0 INTERFERENCES 6.1 There areno known interferenceastthistime. 7.0 SAMPLE HANDLING 7.1 The rabbitliversarereceivedfrozen,and must *bekeptfrozenuntiltheextractioins performed. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof InternalStandards 8.1.1 Preparean intemalstandardof approximately12 ppm 1H,lH,2H,ZHperfluorooc=esulphonicacidtobe added toeach liversample. 8.1.2 Weigh atleast0.1 g of 1H,1H,2H,2H-perfluorooc=esulphonicacidintoa 100 mL volumetricflask.Record theactualweight. 8.1.3 Bringitup tovolume withmethanol,thisisthestockstandarcl 8.1.4 To a 250 mL volumetricflask,add 3 mLs of thestockstandardand bringto volume with MRH-Q water.Calculatetheactualconcentratioonf the standard. actualmg perfluoroctane- sulphonicacid X 0.1 L 3 mL 250 niL actualconcentrationp,pm 8.2 Prepare FC-95 Anion Standards 8.2.1 PrepareFC-95 standardsforthestandardcurve. 8.2.2 Weigh approximately100 mg of FC-95 intoa 100 mL volumetricflask. Record theactualweighl 8.2.3 Bringup tovolume withdiluteacetonitdle. 8.2.4 Dilutethesolutiownithdiluteacetonitdl1e:10 fora solutioonf approximately100 ppm. Dilutethissolution1:10 withdiluteacetonitrifloer a solutionof approx.10 ppm. 8.2.5 Use the 10 ppm solutionto make worldng standardswithvaluescloseto 5.0 ppm, 1.0ppm and 500 ppb. 8.3 Prepare Beef Liver Homogenate to Use for Standards 8. 3. 1 Weigh 40 g of Bovineliverintoa 250 mL Nalgenebotdecontaining 200 mLs Nfilli-wQater.Grind toa homogenous solution. 8.3.2 Add I mL of thesolutionto a 15 mL centrifugteube.Preparea totaolf eight I mL aliquotosf thesolutionin 15 mL centrifugteubes.Be suretorr,suspend solutionby shakingitbetween aliquots. 0()00(1;. 3 8.3.3 SpikesevenoftheImL aliquowtisththefollowianmgountsofworking standardsinstep9.12of theprocedure.One I mL aliquotservesas the blank. Worldng Standard UL (Appmximate Conc.) 500 ppb 500 ppb 500 ppb 500 ppb 1 pm 5 ppm 5 ppm - 100 2M 300 400 5E ---Iu 300 Approximatefinalconcentratioonf FC-95 inliver Blank 0.292 ppm 0.584 ppm 0.877 ppm 1.168 ppm 2.924ppm 5.848 ppm 8.772 ppm 8.4 Calculatetheactualvalueof thestandards: uL of standardx concentratio(ninppm) = finacloncentratio(nppm) 171 mg liveF/Iml homogenate ofFC -95inliver *Average weightof bovineliverin solutionasdeterminedby weighing InaL homogenatesof40 mg liverin 200 mL of MMI-Q water.The amount of FC-95 isreportedas equivalentosf FC-95 potassiumsalt. 8.5 Calibration 8.5.1 ExtractthespikedbeefEver homogenate following9.13to9.23of this method. Use thesestandardstoestablisyhour curve on themass spectrometer. 8.5.2 Alternativelay,standardcurve may be generatedusingratiosof responses of theperfluorooctansulfonaatneionand theinternalstandardanionversus concentratioonf theperfluorooctanesulfonatneion. 8.6 Storage Conditions for Standards 8.6.1 New standardsarepreparedwith each analysisS.tandardsarestoredin coveredplasticentrifugteubesuntiltheanalysison themass spectrometer isperformed. 8.7 Storage Conditionsfor Standards 8.7. 1 Beef liverhomogenates may be frozenafterpreparation. 9.0 PROCFDIJRIF.S 9.1 Obtain frozenliversamples.In spenttissuen,otethattheliverhasnotbeen packaged with othertissues. 9.2 Use a dissectinsgcalpeland cutoffapproximately1 g of liver. 9.3 Weigh thesampledirectliyntoa taredplastigcrindingtube. 9.4 Record theEverweightinthestudynotebook. 9.5 Put a labelon thevialwiththestudynumber, weight,rabbitD:),dateand analyst initials. c (.100 (1; 4 9.6 9.7 9.8 9.9 9.10 9.11 9.12 9.13 9.14 9.15 9.16 9.17 9.18 9.19 9.20 9.21 9.22 9.23 9.24 9.25 Add 2.5mLs water. Grind thesample.Put thegrinderprobe inthesample and grindforabout 2 minutes,untilthesample isa homogeneous solutionwithno largechunks. Rinse theprobe offintothesample with 2.5mLs water usinga pipet. Take thegrinderapartand cleanitwithmethanolaftereach sample.Follow AMDT-EP-22. Cap thesample and vortexfor15 seconds. Pipet1 niL intoa 15 mL centrifugteube.Label thecentrifugteubewiththeidentical informationas thegrindingtube.(SeeAMDT-M-4 Worksheet fordocumenting the remainingsteps.) Spike thebeefliverhomogenateswiththeappropriataemount of FC-95 standard as describedin 8.3. Spike thesamplesand beefliverhomogenates with 100 uL of internasltandard. Add 1 mL of thesodium carbonate/sodiubmicarbonatebufferand I mL ammonium acetate. Using an analyticaplipet,add 5 mL ethylacetate. Cap thesample and vonex 20 to30 seconds. Put them intheshakerfor20 niin. Centrifugefor20 to25 minutes,untilthelayersarewellseparatedS.etthepower on thecentrifugeto25. Remove 4 mLs of thetoporganiclayertoa fresh15 mL centrifugteubewitha 5 mL graduatedglasspipet.Transferthelabeltothefreshtube. Blow thes=ple down on theanalyticaelvaporatortoneardrynesswithnitrogen, approximately30 to40 minutes. Bringtheremainingsampleup inI mL diluteacetonitriwliethan analyticaplipel Vortex 15 seconds. Transferthesample toa 3 mL syringeA.ttacha 0.2@= nylon mesh filtera,nd filter thesample intoa freshcentrifugteubeor a autovialL.abel thetubeor vialwiththe studynumber and animalnumber. Cap and holdforanalysisby electrospramyass spectroscopy. Complete ANMT-M-4 worksheetand attachtopage of studynotebook. 10.0 VALIDATION 10.1 QualityControl- not applicable 10.2 Precisionand Accuracy- not applicable 10.3 Other ValidationParameters-not applicable 11.0 DATA ANALYSIS 11.1 None 12.0 ATTACHMENTS 12. 1 WorksheetAMDT-M-4 13.0 R FEREN ES 13. 1 AMDT-EP-22 RoutineMaintenanceofLJ]tra-TurrTa-x25 14.0 REVISIONS Revision Number Reason forChanee Revision Date oooo(;(5 Study - Sample Number set Rl;inkT-iver Worksheet AMDT-M-4 FC-95 approx0.5ppm actual PPM #W@-- I(IniiT, 2nO uT. InO iiT. 400 tiT, - FC-95 approx I ppm actual ppm #W inO iiT. FC-95 approx.5 ppm actual PPM #W Date and InitialfsorStd. ?nniiT. 100 uT. qtntiv TilimberWlpi& tht-oricrinnwlorkqh"t iglnentecoincinince.,r,nany, T.ivt-FrxtrAr-hnnPmee PinetImT. nfT.iver1,;nliition Pinet100 iiTn.f 17 nnm Tntt-rnSatlnn&rd Vnrtex.Ii -,L-r Pim,tI mT.nf 71;0nnrn Ammoniiim Art-t:it#-. PinetI mT@ of0-25NarO./O 75M NaT4CO. Riiffer PinetISrnT.of F@thylAcetqtL- vnrtex ?o in qer I;h;tk'-2Ln-min r-entrifij?u0y-e75 min Remnyt-n 4 mT. gliniintf nroanirI;ivt-r 'Blowclnwntn ne;idrrvnesq(<O75 mL) withN. AciciI m- of 1-1 Ac-ptnnitrile/14.0 TN# ynrtpxIS-qelr Filteiriqin2g Ice B-D!;vrincrweitha 0 ?itmSRI filttrinto1a5 mT. ntitn-qamnviwc- r)ntf,Initinlq oooo(;7 6 3M EnvironmentalLaboratorv Method Analysis of Fluoride Using the Skalar Segmented Flow Analyzer With Ion SelectiveElectrode Method IdentiricatiNounmber: AMDT-M-8 Adoption Date: RevisionNumber: 0 RevisionDate: None Author Deb Wright CynthiaWeber Appmved By: ?p up LLenaede@era'@-dr@ Date QualityAssurance Date Software:IBM MS Word, 6.0 AffectedDocuments: AMDT-EP-26, Operationand MaintenanceoftheSkalarSegmented Flow Analyzer 0()OO(SS 1.0 SCOPE 1. 1 This method isfortheanalysisforfluoridet,hermallyextractedfrom samplesusing theDohrmann DX2000 (AMDT-M- 1),and coliccteidnTISAB foranalysiwsithan IonSelectiveElectrode(ISE).The analysisisperformedusingtheSkalar Segmented Flow Analyzerwith ISE. 1.2 Samples can be tissuess,erum,biologicamlaterialo,r othermaterialsextracteodn theDohrmann. 2.0 KEYWORDS 2.1 Skalar,segmented flow,fluoride. 3.0 PRECAUTTONS 3.1 Followstandarldaboratorsyafetpyractices. 4.0 SUPPLTES AND MATERTALS 4.1 Supplies 4.1.1 Sample cups,4 mL plasticups withcaps 4.1.2 Autopipetso,xfordor equivalenwtithplastictips 4.1.3 Polypropylenevolumetricflasks,100 mL 4.1.4 Cartridgecomponents,refertotheSkalarMethods forcomponents and part numbers. 4.1.5 Sample prefilterEsv,ergreen 4.2 Reagents 4.2.1 Brij35, 30% S.F.A.S.Detergent 4.2.2 TISAB 11buffersolutionP:urchaseTISAB IIfrom Orion.To I liteorf TISAB 11add 2.5mL or 100 ppm fluoridseolutionand ImL Brij. 4.2.3 SamplerrinsingsolutionD:iluteTISAB 111:1 withMilli-Qwater. 4.2.4 Nitricacidsolutiofnordecontamination1,N (labgrade):Slowly add 64 mLs concentratenditriaccid(HNO 3) to250 mLs of Milli-Qwater.Bring thevolume up toIL withMilli-Qwater. 4.3 Standards 4.3. 1 Stock solution1,00 ppm F:purchasedfrom Orion. 4. 3.2 Intermediatsetandard,10 ppm: Dilute10 mLs of stocksolutionto 100 mLs withMilli-Qwater.Use polypropylenevolumenic flasks. 4.3.3 Working standardM:ake up thefollowingworking standardsby addingthe volumes ofintermediatoer stockstandardindicatedon thetable,using oxfordorpumpmate pipetst,o50 mLs ofTISAB and dilutintgo 100 mLs withMilli-Qwater. Working Standard mLs of Stock Standard mLs of IntermediatSetandard 0.015 ppm 0.15 0.03 ppm 0.3 0.0 ppm 0.6 0.09 ppm 0.9 0.12 ppm 1.2 0.15 ppm - 1.5 0.3 ppm 0.3 - 0.6 ppm 0.6 2 ()(100"9 1.2ppm 1.2 1.5ppm 1.5 5.0 EQUIPMENT 5.1 SkalarSegmented Flow Auto Analyzer Sansp'"System equipped with ISE 6.0 TNTERFERENCES 6.1 High concentrationosfalkalinitcyh,loridep,hosphate,sulfateor ironcan cause interferences. 7.0 SAMPLE HANDLTNG 7.1 Samples should be storedinpolyethylenebottlesS.amples should be analyzed within30 days. 8.0 CALIBRATTON AND STANDARDTZATTON 8.1 Preparation of CalibrationStandards 8. 1.1 Prepare calibratiosntandardsas in section4.3. 8.2 Calibration 8.2. 1 The standardsareanalyzedatthebeginningof therun. 8.3 Storage Conditions for Standards 8.3.1 Standardsarestoredincapped polypropylenevolumetricflasks.New standardsarepreparedata minimum ofevery sixmonths, or as necessary. 9.0 PROCEDURE 9.1 Start Up Procedure 9.1.1 Clamp down the pumpdecks, airbarsand sampler-pump tubing. 9.1.2 Put thefluorideelectrodesintheelectrodechamber. 9.1.3 Turn on thepower of the sampler,pumps, offsetpotentiometerand headng bath. 9.1.4 Put the reagent-lineisntheappropriatebottles. 9.1.5 Turn on theinterfacec,omputer, displayand printerM.ake sure you turn on the interfacebefore the computer. 9.1.6 Letthesystemstabilifzoerapproximatel3y0 minutes. 9.2 Startinga Run 9.2.1 Createa sample tableby selectingFILES, TABLE, and CREATE, type in the name of the filea,nd pressENTER. 9.2.2 Printthe sample table,insertedin thesystem tableby pushing ESC, PRINT, GROUP 1.This willprinttheentirerun. 9.2.3 Dial thesampler settingstothe appropriatenumber of samples,number of seconds forsample wash, and number of seconds forthesample. 9.2.4 Fillthe sample traywith thestandards,samples,washes and driftsr.W and FW/RUNOUT cups on thesampler do not need tobe filled. 9.2.5 Set thebaseline. 3 00 9.2.S. ISelectGRAPHICS, REAL TIME. Ifyou cannotgetreal-timey,ou may be intheData HandlingPanel.Switch totheAnalysisPanel by selectinCgONTROL PANEL and pushingF7. 9. 2.5.2 Use thesmallscrewdriverfortheoffsetpotentiometetrosetthe baselineA.djustthebaselineuntilitisapproximately3/4inchfrom thebottom of thescreen. 9.2.5..3Checkthehigheststandardand adjustthegain,ifnecessary,with theinterfacsecrew #3. 9.2.6 Go toCONTROL PANEL, and toanalysispanel.Deselecttheanalysisthat willnot be run.(Selector deselectanalysisby pressingENTER.) PressTab toreturntotheAnalysisPanel. 9.2.7 Pressthespacebartobringup thelocalmenu. 9.2.8 SelectSTART tostartheanalysis. 9.2.9 Type yourID (initialtsh)e,sampletablewhich you createdunder9.2.1(or pressENTER forchoices)c,hoose runningwithorwithoutthesystem table and selectSTART ANALYSIS. 9.2. 10 Afterstartintghesoftware,starthesampler.Make surethatthesampleris settotherightnumber of samplesand thatthesample/wash/aitrimesare OK. 9.2.11 SelectGRAPHICS, REAL TIME toview theprogressof theanalysis. 9.3 Loading and Printing the Data-File 9.3.1 Go toCONTROL PANEL, pressthespacebartobringup thelocalmenu and selecLtOAD. SelectAUTOCALCULATION and enterthefilename(or highlightthefiletobe printedand pressENTER). 9.3.2 To view thecalibratiocnurve,go toGRAPHICS, CALIBRATION CURVE. 9.3.3 To printthehighlevelcurve,push PRINT SCREEN. 9.3.4 To printthelow levelscreen,push ESC to getoutof graphics.Select SETTINGS. Change themax y valuetoapproximately900.Go toCAL CURVE and pressESC, and Enter.PressPRINT SCREEN. 9.3.5 ReturntoS=NGS and changethemax valueback to4095,go toEDIT, pressENTER and PRINT SCREEN toprintsample peaks. 9.3.6 To printtheresultgso toCONTROL PANEL, SPACEBAR, OUTPUT, OUTPUT. SelectPRINTER fortheEpson or PRN fortheLaser. 9.4 Shutdown 9.4.1 Put allthereagent-liniensMilli-Qwater. 9.4.2 Let thesystem rinseforapproximately30 minutes. 9.4.3 Afterthesystem hasrinsedcompletely,turnoffthesampler,pump and offsetpotentiometerT.urn offtheheatingbathon weekends.Leave liquidin the lines. 9.4.4 Take theelectrodoeut and soak in 100 ppm F overnight. 9.4.5 Releasethepump-decks, airbarsand samplerplimp-tubing. 9.4.6 SelectFILES, pressALT F and selecQtUIT toexittheprogram. 9.4.7 On Friday,turnoffthecomputer,displayand interfacfeortheweekend. 10.0 VALIDATION 10.1 QualityControl 10. 1. 1Run a standard(mid tohighconcentratione)very 10 'samplesI.fa significancthange inpeak heightoccurs,only thesamplesbeforethelast acceptablestandardwillbe used.The remainingsampleswillbe reanalyzed. 4 0 0 0 0"1"1 10.2 Precisioannd Accuracy 10.@.1SeeMethoVdalidatRieopnorntumberAMDT-M-8.0.IV 10.3 Other ValidationParameters 10.4 RefertoMethod ValidatioRneportNumber AMDT-M-8.0.Vl 11.0 DATA 11.1 11.2 11.3 11.4 ANALYSIS Calculations 11.1.1The standardcurveisplottedby theSkalarsoftware. 11. 1.2 Allcalculatioanrsedone by theSkalarsoftwarer.' shouldbe 0.995or better. Preparespreadsheettsosununarizedata.Includes=ple volume, weightsusedetc. Writethestudynumber on theprintoutsi,nitiadla,tetheprintouta,nd bindtogether withallpackagedocumentsand placeinthestudyfolderM.ake acopy ofthe summary sheetand tapeintothestudynotebook.Back up alldataand spreadsheets ontostudydiskand backup disks. ElectroniDcata 11.4.1GLP studiesE:lectronidcataiscopiedontotheStudy floppydiskforeach study,andalsodataiscopiedontoa floppydiskthatisstoredinthelab. 11.4.2 OtherstudiesA:lldataiscopiedontoa floppydiskthatisstoredinthelab. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1 AMDT-M-1, ThermalExtractioonfFluorideby Means of a ModifiedDohrmann DX2000 OrganicHalideAnalyzer-Liver 13.2 SkalarMethods,#335,SkalarMethods Manual 13.3 AMDT-EP-26, Operationand Maintenanceof theSkalarSegmentedFlow Analyzer 14.0 REVISIONS Revision Numbe Reason forchange Revision Date 5 1 OKo 0()Oo"v--,b 3M Environmental Laboratory Method "Mermal Extractionof Fluorideby Means ofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer- Serum Method IdentiricatNiuomnber: AMDT-M-14 RevisionNumber: 0 AdoptionDate: RevisionDate:None Author.RichYoungblom Approved by: 00"-i up Leader Date ) ZP L z QualityAssurance Date Software:MS Word 5.la AffectedDocuments: AMDT-M-2 FluoridMeeasurementby Means ofan OrionEA940 ExpandableIonAnalyzer AMDT-EP-3 RoutineMaintenanceofa Modifi"eDdohrmann DX2000 OrganicHalideAnalyzer 1.0 SCOPE. APPLICABLE COMPOUNDS, AND MATRICES 1.1Scope:Thismethod isfortheoperatioonfa Dohrmann DX2000 when itisusedtoextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2ApplicableCompounds: Fluorochemicalosrotherfluorinatecdompounds. 1.3Matrices:Biologicafllwds,particularsleynun 2.0 KEYWORDS 2.1Fluoridef,luorinee,xtractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS 3.1 Glasswareand exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizedgases,propercompressedgashandlingpracticersequired. 3.4Solventbased samplesmay flashm,ay need toallowthem todrydown beforestartinngm. 3.5Potentiabliohazardsdue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLIES AND MATERIALS 4.1Compressed Oxygen, Hydrocarbon free,regulatetdo30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3QuartzglasssampleboatwithTeflonTmtubing,Dohrmann 890-097orequivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012orequivalent. 4.5Orion940999 TotalIonicStrengtAhdjustmentBuffer(TISAB II)orequivalent. 4.6Sample collectiovnialsH,DPE. 4.7 Nfilh-QT1w4ater 4.8Polystyrenepipettes. 4.9ActivatedCharcoal,E.Merck 2005 orequivalent. 4.10Hamilton Syringeor equivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQUIPMENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzer,modifiedforfluorideextraction. 5.2MM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluorideextraction. 5.4 ExcelSpreadsheetv,ersion5.0or greater 6.0 TNTERFERENCES 6.1 Sample sizeislimitedtoapproximately100 @Ll.Thi.smay varyfrom matrixtomatrix. 00007. 2 7.0 SAMPLE T-TANDLING 7.1Samples aretobe handledwithplastipcipettesA. new pipetteistobe used foreach sample. 8.0 CALTBRATION AND STANDARDIZATION 8.1 PreparationofCalibrationStandards 8.1.1The standardrsequiredforeachprojecwtillneedtobe appropriatfeorthatindividuaplroject. Refertoprotocolforthatproject. 8.1.2Typically50-500 ppm FC-95 inmethanol standardsareused. 8.1.3For rabbitserum studiesu,se beefserum asthematrix. 8.2 Calibration-Overview The normal calibratioinsthefluoridceurve(AMDT-M-2). However, ifanoptionalspikedserum curveisrequiredtheprocedurelistebdelow isused. 8.2.1A calibratiocnurvefortheDX2000 isgeneratedby spikingsampleswithknown standards and combusting them usingthesame methods and matrixtype asthesamples tobe tested. 8.2.2Typically,threereplicateosf eachstandardand fiveconcentrationosf standardswillbe spiked. 8.2.3Standardcurvewillbe plottedasMass SpikedF (ug)on thex-axisand StandardMass Recovered F (ug)on they-axis.Generatea regressioncurve and calculatteheequationfortheline and ther2value. 8.2.4Mass SpikedF (ug)= (Amount spikedinmL) x (Conc. ofstandardinppm) x (0.6004)* *FC-95 is60.04% F therefore0.6004isthefactorused toconvertFC-95 to F 8.2.5StandardMass RecoveredF (ug)= (TISAB volume inmL) x (Orionreadinginppm) 8.3Calibration- Procedure 8.3.1StartUp 8.3.1.1Run 2 ormore CleanCycleswhen startinignstrumenetachday. More cleancyclesmay be usediftheprevioussamplescontainedhigh concentrationosf fluoride. 8.3.2Blanks 8.3.2.1Preparesample usingthesame methods and typeof matrixas thetestsample. 8.3.2.2For rabbitstudiesu,se beefserum as thematrix. 8.3.2.3Put serum blank,inDohrmann boat. Combust sample as describedinsection9.0and analyzesample accordingtomethod AMDT-M-2 fortheionselectivelectrodaenalysis. 8.3.2.4For rabbitstudiest,hemeterreadingfora blanksample shouldbe 0.03ppm or lower beforeproceedingwiththecalibratioBnu.m samplesuntilthislimitisreached,oruntilinthe judgement of theoperatorthereadingisstablewithrespecttohistoricarleadings(previous48 hours). 8.3.2.5For non-rabbitstudiest,heblank-readingsshouldreacha predeterminedionconcentration beforeproceedingwiththecalibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwiththesampletoaid combustion. 0 007 3 8.3.3Standard Curve 8.3.3.1Ifbeefserum isfrozen,thaw atleastenough tocompletethestandardcurveanalysisforthe day (=30 mL). 8.3.3.2PipetteI00@iLof beefserum intoDohrmann sample boat. 8.3.3.3StartwiththeloweststandardconcentrationU.sing a Hamilton syringe,ejecta fixed quantityofthestandardon or in thematrix.For rabbitstudiesu,se 4 uL of standardand ejectiton or inthebeefsenun. 8.3.3.4At least3 replicatesshouldbe usedforthelowest standardconcentrationm;ore replicates may be used atthediscretioonftheanalyst. 8.3.3.5Combust thesample as describedin section9.3and analyzeaccordingtoAMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessignificantdliyfferenftrom theothertwo replicatensm, anothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe oldreplicataend thatthenew replicatweillbe used to calculatteheregressiocnurve. 8.3.3.7When allstandardshave been run,calculattehe r2. r2 must be atleast0.95.Ifitisnot at least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshouldbe run when thecombustiontubeor sample matrixis changed. New standardcurvemay alsobe run atthediscretioonf theanalyst. 8.4 StorageConditionsforStandards 8.4.1Storagerequirementsforstandardsaredependenton theindividuasltandardsused. Typically,standardsarestoredatroom temperatureinplastisccrew topbottles. 8.4.2New FC--95 standardsshouldbe preparedatleastonce a month. 9.0 PROCEDURES 9.1TypicalOperating Conditions: 9.1.1Combustion tubetemperature= 950'C. 9.li2Oxygen and Heliwn flow 50 cc/minute. 9.1.3Vaporization/Dryintgime 240 seconds. 9.1.4Bake time = 300 seconds. 9.2StartUp Procedure: 9.2.1Iftheprogram isnotstarteds,tartheEOX program on thePC. 9.2.2Open theSYSTEM SETUP window. 9.2.3Put thefurnacemodule and thecellintheREADY mode. 9.2.4Close the SYSTEM SETUP window*. 9.2.5When theoven has reachedtheREADY temperaturer,un theCLEAN BOAT program foundintheCELL CHECK menu. 9.2.6See ANMT-EP-3 fordetailosftheDohmiann software. 9.3Sample ExtractionProcedure: 9.3.1Open theSAMPLE HATCH and pipette1OOGL of sample intotheBOAT. Itmay be necessarytomix approximately50 mg of charcoalwiththesample toaidcombustion.Ifthisis done,charcoalshouldalsobe mixed inwhileestablishintghebaselineand when generatingthe standardcurve. 9.3.2Close SAMPLE HATCH. 00007ti: 4 9.3.3Add appropriatveolume ofTISAB solutioonr1:1 TISAB:Milli-QTm watermixtureto a labeledsample collectiovnia].Typically0.6mL to15 mL areused. For rabbitstudiesu,se 1.0or 2.0mL of 1:1 TISAB:Milli-QTM watern-dxture. 9.3.4Placethevialso thatthetipoftheCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releasedduringpyrolysimsust bubblethroughtheTISAB. 9.3.5 Run theEOX-WATER program foundintheRUN menu. 9.3.6When theEOX program isfinishedr,emove thecollectiovnialfrom thecombustiontube. 9.3.7IfundilutedTISAB was usedtocollecthesample,add an equalvolume of Milli-QTMwater totheTISAB tomake 1:1 TISAB:Milli-QTm. 9.3.8Rinse theend of thecombustiontubewithMilli-QTMwaterand wipe witha KIMWIPE to remove any TISAB remainingon thetube. 9.3.9Open the sample hatchand remove any remainingash from theboat. Ash can be removed witha cottontippedapplicatoarnd/orvacuumed out.Itmay be necessaryto scrapparticleosffthe bottomwith a spatulaor othersimilardevice.A dropof Milli-QTmwater may be added to the boattoaidintheClean Cycle. 9.3.10Close thehatch. 9.3.11Run theCLEAN BOAT program. 9.3.12Sample isreadyforanalysibsy ion selectiveelectrod(eAMDT-M-2). 9.4 Sample Calculations 9.4.1Use thestandardcurvetocalculattehesample value. 9.4.2Sample Mass RecoveredF (ug)= (TISAB volinmL) x (Orionreadingin12p -intercel2i) (Slope) 10.0 VALTDATION 10.1 QualityControl 10.1.1Daily StartUp Check Samples: Once thestandardcurveisestablishede,ach day of analysisisstartedby analyzingQC samples.The QC samples areto be thesame asthelowest concentratiosnpikedsamplesused togeneratethestandardcurve.Each concentratiomnust be done intriplicautnelessthefirstwo replicateasrewithin20% of thestandardcurve,thena third replicatiesnot necessary. 10.2 Precisionand Accuracy: See method developmentanalysisand sampleanalysisin FluorideNotebooks 2,3,and 5. Precisionand accuracyvarieswhen analyzingsamplesof different matricesand differenrteferencecompounds. 10.3 Other ValidationParameters: NA 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1For thestandardcurve,useregressionanalysisinExcel,version5.0or greater. 11.1.2To calculattehe fluoridceontractioinnthesample,seemethod AMDT-M-2. 5 0000,r 11.2AnalyzingtheData 11.2.1r2mustbe atleas0t.95orgreate"rO.utliermsa"y be excludeidftwo ofthethreereplicates arewithin20% ofeach otherand theoutlierisgreaterthan 200% of the average ofthose two or lessthan 50% ofthe average of thosetwo. Any such outliershouldbe pointedout inthe dataand noted inthe FinalReport along withthereasonitwas consideredan outlier. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1Rosemount Dohmiann DX2000 Organic Halide Analyzer Operator'sManual (Manua.1915349,revisionB, December 1993) 13.2 ANMT-M-2 FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 13.3AMDT-EP-3 RoutineMaintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 REVISIONS Revision Numbe Reason forChangg Revision Date 6 I -@ ..@ I . I Iil---. 9.3 QualityAssurance Unit Statement 0()00"9@!) AttachmentD GLP Study QualityAssurance Statement Study Title: Single-doseIntravenous Pharmacokinetic Study of T-6246 in Rabbits StudyNumber. AMDT-042095.1 Name ofAuditor:KafiRambo This studyhas been hwected by theQualityAssuranceUnit asindicateidnthefollowingtable. 7[befindingswere reportedtothestudydirectoarnd management. InspectioDnates From -T2 11-13-95 11-13-95 Phase FinalReport Date InspectioRneportedto Manag=nt StudyDirector 11-13-95 11-13-95 'QAU Auditor Date 0(.)O("SO ' -.%-. 9.4 Key PersonnelInvolvedin the Study OOOOSJL 3M Environmental Laboratory Key Personnel ThermalextractifonllowbeydanalysuissinOgrionionanalyzer: Jim Johnson Deb Wright RichYoungblom Deann Plummer Thermal extractiofnollowedby analysiussingSkalarsegmentednow analyzerwithionselectievleectrode: Jim Johnson Deb Wright RichYoungblom Deann Plummer Documentationand Reporting: Jim Johnson RichYoungblom QualityAssuranceUnit: GaleVan Buskirk CynthiaWeber KariRambo 00 .,.. 9.11 Data .@ --. 44,Mi- 0()0083 9.11.1Summary and raw data;ug F-inwhole liveras determinedby thermalextractiofnollowedby analysisusing Orion ion analyzer. 0000811 Summary ofCombustion Data -Liver AMDT-042095.1, HWI 6329-159 As Referenced inFinalReport section6.0DATA ANALYSIS Total@LgFluorideinWhole Liver Mean per Dose Group per Day Sacrifised* 119 Std.Dev. ControlGroup, Day 15** 27.1 3.1 ControlGroup, Day 28 38.7 16.3 100 mg/kg dose (T6246),Day 15 37.4 7.9 (0.12mg/kg)*** 100 mg/kg dose(T6246),Day 28 29.6 1.3 (0.12mg/kg)*** 500 mg/kg dose(T6246),Day 15 123.1 14.0 (0.60mglkg)*** 500 mgtkg dose (T6246),Day 28 100.5 5.9 (0.60mg/kg)*** *Calculatedas a mean triplicatseamplesfrom each oftwo male and two femalerabbits. **Only two liversanalyzedfrom thisgroup. ***Testmaterialisa 0.12% solutionofFC-95, actualdose inparenthesis. OoOO85 FCOS ID liverblank-1 livebrlank-2 livebrlank-3 F54075-1 F54075-2 F54075-3 F54081 -1 F54081-2 F54081-3 F54105-1 F54105-2 F54105-3 F54107-1F54107-2 F54107-3 F54074-1 F54074-2 F54074-3 F54085-1 F54085-2 F540853 LiverBik-I LiverBik-2 LiverSpk-1 LiverSpk-2 LiverSpk-3 LiverSpk-4 LiverSpk-5 blank 1 blank2 spike-1 spike-2 spike-3 spike-4 spike-5 spike-6 bik1 blk2 spkl spk 2 spk 3 spk 4 F54082-1 F54082-2 F54082-3 F54111-1 F54111-2 F54111-3 % rcvry 40% 45% 40% 39% 54% 91% 106% 118% 97% 138% 96% 119% 113% 106% 103% Actual Average ppm F- ppm Finliver inliver (WNV) (WNV) 0.776 0.500 0.480 0.401 0.523 0.412 0.313 0.308 0.448 0.387 0.404 0.267 0.431 0.339 0.319 1.34 0.542 0.780 0.459 0.350 0.265 0.336 0.393 0.312 0.272 0.407 0.637 0.437 0.185 0.553. 0.469 0.389 0.418 0.660 0.528 0.334 1.26 1.11 1.15 1.04 1.65 1.06 0.530 0.264 1.15 1.14 0.979 1.11 1.22 1.25 1.23 1.21 1.21 1.11 1.17 1.17 Whole TotalF-in liver liver whole burned weight liver (grams)(grams) (ug) 0.113 0.117 0.131 0.131 60.3 0.120 60.3 24.8 0.1.32 60.3 0.125 80.6 0.116 80.6 31.2 0.108 80.6 0.151 86.4 0.105 86.4 29.3 0.117 86.4 0.107 80.6 0.135 80.6 62.9 0.129 80.6 0.123 82.9 0.144 82.9 27.9 0.111 82.9 0.149 81.0 0.149 81.0 33.0 0.101 81.0 0.097 0.148 0.110 0.145 0.156 0.142 0.125 0.097 0.148 0.110 0.145 0.156 0.142 0.125 0.137 0.153 0.161 0.157 0.150 0.164 0.141 0.160 89.0 0.163 89.0 109.4 0.156 89.0 0.121 83.8 0.115 83.8 97.7 0.136 83.8 Dosage (mg/kg) 0.0 0.0 0.0 0.0 0.0 0.0 500 Soo Page 1 RPT1 59L.XLS fi()00@ FC95 Actual Average Whole TotalF- in ppm F- ppm F- liver liver whole ID % inliver inliver burned weight liver Dosage rcvry (W/W) (WNV) (grams)(grams) (ug) (mg/kg) F54079-1 F54079-2 1.17 0.157 83.1 1.20 1.18 0.142 83.1 97.9 Soo F54079-3 1.16 0.141 83.1 F54116-1 F54116-2 1.36 0.154 72.0 1.33 1.35 0.140 72.0 97.1 500 F54116-3 1.36 0.157 72.0 F54112-1 F54112-2 1.44 0.134 97.8 1.55 1.46 0.158 97.8 142.9 Soo F54112-3 1.40 0.146 97.8 F54076-1 F54076-2 1.69 0.139 78.1 1.81 1.76 0.124 78.1 137.4 500 F54076-3 1.78 0.114 78.1 F54113-1 F54113-2 1.24 0.145 91.5 1.33 1.33 0.123 91.5 121.7 Soo F54113-3 1.42 0.117 91.5 F54084-1 1.58 0.134 66.4 F54084-2 1.73 1.66 0.140 66.4 110.4 500 F54084-3 1.68 0.120 66.4 F54098-1 1.46 0.135 79.0 F54098-2 1.39 1.49 0.147 79.0 117.3 500 F54098-3 F54087-1 F54087-2 1.61 0.125 79.0 0.400 0.130 83.7 0.346 0.376 0.131 83.7 31.5 100 F54087-3 0.382 0.135 83.7 F54088-1 1.33 0.134 84.3 F54088-2 0.485 0.765 0.148 84.3 64.4 100 F54088-3 0.482 0.134 84.3 LIVER BLK 1 0.259 0.164 LIVER BLK 2 0.132 0.158 LIVER SPK 1 89% 1.05 0.128 LIVER SPK 2 111% 1.30 0.129 LIVER BLANK 0.243 0.140 LIVER SPIKE 3 111% 1.27 0.133 liverblank 0.355 0.111 F54089-1 F54089-2 0.398 0.153 71.8 0.394 0.394 0.157 71.8 28.3 100 F54089-3 0.391 0.147 71.8 F54099-1 F54099-2 0.502 0.139 82.1 0.504 0.483 0.140 82.1 39.7 100 F54099-3 0,444 0.158 82.1 F54114-1 F54114-2 0.337 0.157 81.0 0.372 0.359 0.136 81.0 29.0 100 F54114-3 0.367 0.155 81.0 F541 09-1 F54109-2 0.383 0.133 74.0 0.387 0.400 0.134 74.0 29.6 100 F54109-3 0.431 0.154 74.0 F54080-1 F54080-2 0.460 0.132 75.4 0.475 0.356 0.132 75.4 26.8 100 F54080-3 0.132 0.103 75.4 . Page 2 RPT159L.XLS c;0 0 FC95 ID F54092-1 F54092-2 F54092-3 F54106-1 F54106-2 F54106-3 Blank Liverspike 1 Liverspike2 Liverspike3 Liverspike4 Liverspike5 Liverspike6 liverblank-1 liverblank-2 liverblank-3 liverspike 63-1 liverspike63-2 liverspike63-3 liverspike63-4 F54088-1 F54088-2 F54088-3 liverblank-4 liverspike 63-1 liverspike 63-2 liverspike63-3 liverspike63-4 Actual Average ppm F- ppm F- % inliver inliver rcvry (WNV) (WNV) 0.537 0.463 0.478 0.433 0.639 0.665 0.634 0.598 0.181 82% 1.09 81% 0.755 85% 0.897 76% 1.47 66% 1.36 70% 1.40 0.549 0.454 0.278 103% 1.09 96% 1.11 159% 1.69 101% 1.41' 0.485 0.655 0.544 0.491 0.292 117% 1.30 89% 0.972 89% 0.922 90% 0.910 Whole TotalF- in liver liver burned weight whole liver (grams) (grams) 0.117 78.4 (ug) 0.145 78.4 37.4 0.138 78.4 0.134 82.3 0.125 82.3 52.2 0.121 82.3 0.133 0.113 0.158 0.144 0.156 0.147 0.152 0.147 0.133 0.110 0.143 0.132 0.143 0.108 0.145 84.3 0.123 84.3 45.8 0.132 84.3 0.123 0.136 0.139 0.147 0.149 Dosage (mg/kg) 100 100 100 RPT159L.XLS Page 3 00()088 9.11.2Summary and raw data;ug F-inwhole liveras determinedby thermalextractiofnollowedby analysisusing Skalarsegmented flow analyzerwithion selectivelectrode. oooos..) RE: 6329-159 LIUER SRMPLES RMOT 42095.1 Date of Rnaigsis:5-23 and 5-24-95 Rnalyst: ODW The samples are burned in the Bohrman at 950 C using between 0.1 and 0.2 grams of the liuer.The gas iscollectedin 2.0 mL of 1:1TISRB/Milli-Owater. The samples are then analyzed on a Skalar Segmented Flow Rnalyzer using the Ion SpecificElectrode (ISE)Method. TISRB buffer isadded to each sample as itproceeds through the system. The sample then goes through a heated mixing coil before the potentialbetween the ion selectiueelectrodeand the reference electrode ismeasured. The signalisamplifiedand relatedto the fluorideconcentration. The instrument was calibratedin the ranges of 0.015 - 0.15 ppm and 0.15 - 1.50ppm fluoride.The standard curue for the high range was plotted using the inuerse logarithm option.The standard curue for the low range islinear.Rilstandards and samples were then calculatedby the Skalar software using these curues. Rilresultsbelow 0.0001 ppm appear on the raw data as R qualitycontrolstandard was analyzed euery 10 samples to check for accuracy and drift. Raw data istaken from the appropriate calibratedrange of the Skalar printoutand summarized on an Excel spreadsheet. The finalresultsare adjusted for the collectionuolume and any subsequent dilutions. oooo9o SUMMARY LIVER -------------------------------------------------------- AMDT of 6329-159 SAMPLES 42095.1 GROUP I Dose Level: 0 F54075-1 F54075-2 F54075-3 F54081-1 F54081-2 F54081-3 F54105-1 F54105-2 F54105-3 F54107-1 F54 10'1'-2 F54107-3 F54074-1 F54074-2 F54074-3 F54085-1 F54085-2 F54085-3 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.45 0.05 0.03 0.02 0.02 0.02 0.02 0.02 0.06 2.0 0.1311 0.50 60.2500 30.06 2.0 0.1197 0.55 0.52 60.2500 33.32 27.4 2.0 0.1318 0.31 60.2500 18.93 2.0 0.1246 0.32 80.597 25.87 2.0 0.1161 0.36 0.33 80.597 29.30 26.8 2.0 0.1075 0.31 80.597 25.34 2.0 0.1507 0.25 86.35 21.54 2.0 0.1053 0.46 0.36 86.35 39.53 31.2 2.0 0.1173 0.38 86.35 32.39 2.0 0.1069 8.38 80.649 675.67 2.0 0.1351 0.75 3.19 80.649 60.53 257 2.0 0.1286 0.43 80.649 34.37 2.0 0.1227 0.37 82.895 31.08 2.0 0.1441 0.25 0.34 82.895 21.05 28.3 2.0 0.1110 0.40 82.895 32.86 2.0 0.1485 0.23 80.95 18.97 2.0 0.1490 0.30 1.81 80.95 23.90 45.8 2.0 0.1005 1.17 80.95 94.40 GROUP4 Dose Level: 100 mgtkg F54087-1 F54087-2 F54087-3 F54088-1 F54088-2 F54088-3 F54092-1 F54092-2 F54092-3 F54106-1 F54106-2 F54106-3 F54088-1 F54088-2 F54088-3 F34089-1 F54089-2 F54089-3 F54099-1 F54099-2 F54099-3 F54114-1 F54114-2 F54114-3 F54109-1 F54109-2 F54109-3 0.04 0.03 0.03 0.11 0.05 0.05 0.06 0.05 0.04 0.06 0.06 0.04 0.05 0.05 0.04 0.03 0.03 0.03 0.04 0.04 0.05 0.03 0.03 0.03 0.03 0.03 0.03 2.0 0.1301 2.0 0.1314 2.0 0.1351 2.0 0.1339 2.0 0.1476 2.0 0.1344 2.0 0.1169 2.0 0.1453 2.0 0.1381 2.0 0.1336 2.0 0.1249 2.0 0.1213 2.0 0.1448 2.0 0.1228 2.0 0.1317 2.0 0.1532 2.0 0.1568 2.0 0.1474 2.0 0.1386 2.0 0.1403 2.0 0.1575 2.0 0.1573 2.0 0.1359 2.0 0.1552 2.0 0.1326 2.0 0.1339 2.0 0.1536 HWI-1 59L.SUM 0.59 0.47 0.51 1.70 0.66 0.68 0.96 0.69 0.60 0.85 0.90 0.71 0.72 0.89 0.63 0.42 0.41 0.39 0.56 0.53 0.59 0.39 0.38 0.40 0.38 0.38 0.45 0.52 1.01 0.75 0.82 0.75 0.41 0.56 0.38 40.30 83.71 83.71 83.71 84.26 84.26 84.26 78.36 78.36 78.'36 82.301 82.301 82.301 84.26 84.26 84.26 71.796 71.796 71.796 82.143 82.143 82.143 80.966 80.966 80.966 73.958 73.958 73.958 49.42 39.24 42.51 143.47 55.26 57.30 75.61 54.15 46.75 70.35 74.33 58.76 60.63 75.07 53.49 29.81 29.12 28.15 46.35 43.21 48.29 31.19 31.10 32.14 28.45 28.17 33.13 43.7 85.3 58.8 67.8 63.1 29 45.95 31.5 29.9 Page 1 0 (11 SUMMARY of 6329-159 LIVER SAMPLES AMDT 42095.1 F54080-1 F54080-2 F54080-3 GROUP5 Dose Level: 500 mgtkg F54082-1 F54082-2 F54082-3 F54111-1 F54111-2 F54111-3 F54079-1 F54079-2 F54079-3 F54116-1 F54116-2 F54116-3 F54112-1 F54112-2 F54112-3 F54076-1 F54076-2 F54076-3 F54113-1 F54113-2 F54113-3 F54084-1 F54084-2 F54084-3 F54098-1 F54098-2 F54098-3 0.03 0.03 ND 0.12 0.12 0.11 0.09 0.08 0.10 0.11 0.11 0.10 0.13 0.12 0.13 0.12 0.15 0.13 0.15 0.15 0.13 0.12 0.11 0.11 0.14 0.16 0.13 0.13 0.14 0.13 ..... 2.0 0.1322 --------0.46 75.42 34.91 2.0 0.1323 0.50 0.32 75.42 37.74 24.2 2.0 0.1028 ND 75.42 ND 2.0 0.1595 1.44 2.0 0.1626 1.48 2.0 0.1558 1.47 2.0 0.1207 1.41 2.0 0.1147 1.34 2.0 0.1360 1.50 2.0 0.1571 2.0 0.1420 1.52 2.0 0.1406 1.46 2.0 0.1541 1.71 2.0 0.1403 1.68 2.0 0.1570 1.68 2.0 0.1340 1.82 2.0 0.1583 1.84 2.0 0.1458 1.79 2.0 0.1385 2.22 2.0 0.1243 2.43 2.0 0.1136 2.35 2.0 0.1449 1.65 2.0 0.1227 1.85 2.0 0,1171 1.94 2.0 0.1339 2.09 2.0 0.1402 2.24 2.0 0.1200 2.22 2.0 0.1346 1.93 2.0 0.1469 1.88 2.0 0.1253 2.12 88.965 128.40 1.46 88.965 -131.75 130 88.965 130.42 83.823 118.48 1.42 83.823 112.69 119 83.823 125.36 83.117 121.58 1.48 83.117 126.67 123 83.117 121.07 71.962 122.82 1.69 71.962 121.15 122 71.962 120.73 97.824 177.98 1.82 97.824 180.45 178 97.824 175.25 78.058 173.59 2.33 78.058 189.65 182 78.058 183.60 91.52 151.08 1.81 91.52 169.17 166 91.52 177.26 66.42 138.90 2.18 66.42 148.76 145 66.42 147.23 78.95 1.98 78.95 148.33 156 78.95 167.23 HWI-159L.SUM Page 2 1995-06-13 10:27 OutPut of: 950523AI Operator : DDW Date of the Analysis 1995-05-23 10:18 Analysis FileName C:ISKALARkDATAIHWIDATAkLIVERS\950523AI HWI-159L.XLS I Tracer 2 Ddft 3 Wash 4 Standard 1 5 Standard 2 6 Standard 3 7 Standard 4 8 Standard 5 9 Standard 6 10 Standard 7 11 Standard 8 12 Standard 9 13 Standard 10 14 Drift 15 Wash 16 BLK 1 17 BLK 2 18 BLK 3 19 F54075-1 20 F54075-2 21 F54075-3 22 F54081-1 23 F54081-2 24 F54081-3 25 F54105-1 26 Drift 27 Wash 28 F54105-2 29 F54105-3 30 F54107-1 1.50 1.50 0.015 0.03 0.06 0.09 0.12 0.15 0.30 0.60 1.20 1.50 1.50 1.50 1.47 98% 1.48 98% 0.00 0.02 125% 0.03 85% 0.06 100% 0.09 102% 0.12 99% 0.15 103% 0.29 95% 0.61 102% 1.24 103% 1.46 97% 1.53 102% 0.00 0.05 0.04 0.04 0.03 0.03 0.02 0.02 0.02 0.02 0.02 1.53 102% 0.00 0.02 0.02 0.45 2.0 0.1332 0.79 2.0 0.1172 0.60 2.0 0.1313 0.60 2.0 0.1311 0.50 60.2500 30.06 2.0 0.1197 0.55 60.2500 33.32 2.0 0.1318 0.31 60.2500 18.93 2.0 0.1246 0.32 80.597 25.87 2.0 0.1161 0.36 80.597 29.30 2.0 0.1075 0.31 80.597 25.34 2.0 0.1507 0.25 86.35 21.54 2.0 0.1053 0.46 86.35 39.53 2.0 0.1173 0.38 86.35 32.39 2.0 0.1069 8.38 80.649 675.67 Page I HWI-1 59L.XLS 31 F54107-2 0.05 32 F54107-3 0.03 33 F54074-1 0.02 34 F54074-2 0.02 35 F54074-3 0.02 36 F54085-1 0.02 37 F54085-2 0.02 38 Drift 1.50 1.52 101% 39 Wash 0.00 40 F54085-3 0.06 41 BLK 1 0.02 42 BLK 2 0.03 43 SPK 1 0.15 44 SPK 2 0.10 45 SPK 3 0.11 46 SPK 4 0.09 47 SPK 5 0.12 48 SPK 6 0.09 49 BLK 1 0.04 50 Ddft 1.50 1.53 102% 51 Wash 0.00 52 BLK 2 0.02 53 SPK 1 0.11 54 SPK 2 0.10 55 SPK 3 0.10 56 SPK 4 0.11 57 F54082-1 0.12 58 F54082-2 0.12 59 F54082-3 0.11 60 F541 11-1 0.09 61 F54111-2 0.08 62 Drift 63 Wash 1.50 1.51 101% 0.00 64 F54111-3 0.10 65 F54079-1 0.11 66 F54079-2 0.11 2.0 0.1351 0.75 80.649 60.53 2.0 0.1286 0.43 80.649 34.37 2.0 0.1227 0.37 82.895 31.08 2.0 0.1441 0.25 82.895 21.05 2.0 0.111 0.40 82.895 32.86 2.0 0.1485 0.23 80.95 18.97 2.0 0.149 0.30 80.95 23.90 2.0 0.1005 1.17 80.95 94.40 2.0 0.0971 0.50 2.0 0.1479 0.39 2.0 0.1096 2.67 2.0 0.1446 1.36 2.0 0.1556 1.39 2.0 0.1416 1.21 2.0 0.1246 1.97 2.0 0.1369 1.32 0.004 0.004 0.004 0.004 0.004 0.004 63.00 63.00 63.00 63.00 63.00 63.00 2.0 0.153 0.58 2.0 0.1609 0.27 2.0 0.1568 1.37 2.0 0.1502 1.35 0.004 0.004 63.00 63.00 2.0 0.1636 1.17 0.004 63.00 2.0 0.1412 1.51 0.004 63.00 2.0 0.1595 1.44 88.965 128.40 2.0 0.1626 1.48 88.965 131.75 2.0 0.1558 1.47 88.965 130.42 2.0 0.1207 1.41 83.823 118.48 2.0 0.1147 1.34 83.823 112.69 2.0 0.136 1.50 83.823 125.36 2.0 0.1571 1.46 83.117 121.58 2.0 0.142 1.52 83.117 126.67 Page 2 HWI-1 59L.XLS 67 F54079-3 68 F54116-1 69 F54116-2 70 F54116-3 71 F54112-1 72 F54112-2 73 F54112-3 74 Drift 75 Wash 76 F54076-1 77 F54076-2 78 F54076-3 79 F54113-1 80 F54113-2 81 F54113-3 82 F54084-1 83 F54084-2 84 F54084-3 85 F54098-1 86 D(ift 87 Wash 88 F54098-2 89 F54098-3 90 F54087-1 91 F54087-2 92 F54087-3 93 F54088-1 94 F54088-2 95 F54088-3 96 Drift 97 Wash 0.10 0.13 0.12 0.13 0.12 0.15 0.13 1.50 1.48 99% 0.00 0.15 0.15 0.13 0.12 0.11 0.11 0.14 0.16 0.13 0.13 1.50 1.49 99% 0.00 0.14 0.13 0.04 0.03 0.03 0.11 0.05 0.05 1.50 1.49 99% 0.00 2.0 0.1406 1.46 83.117 121.07 2.0 0.1541 1.71 71.962 122.82 2.0 0.1403 1.68 71.962 121.15 2.0 0.157 1.68 71.962 120.73 2.0 0.134 1.82 97.824 177.98 2.0 0.1583 1.84 97.824 180.45 2.0 0.1458 1.79 97.824 175.25 2.0 0.1385 2.22 78.058 173.59 2.0 0.1243 2.43 78.058 189.65 2.0 0.1136 2.35 78.058 183.60 2.0 0.1449 1.65 91.52 151.08 2.0 0.1227 1.85 91.52 169.17 2.0 0.1171 1.94 91.52 177.26 2.0 0.1338 2.09 66.42 138.90 2.0 0.1402 2.24 66.42 148.76 2.0 0.12 2.22 66.42 147.23 2.0 0.1346 1.93 78.95 152.03 2.0 0.1469 1.88 78.95 148.33 2.0 0.1253 2.12 78.95 167.23 2.0 0.1301 0.59 83.71 49.42 2.0 0.1314 0.47 83.71 39.24 2.0 0.1351 0.51 83.71 42.51 2.0 0.1339 1.70 84.26 143.47 2.0 0.1476 0.66 84.26 55.26 2.0 0.1344 0.68 84.26 57.30 Page 3 1995-06-13 10:27 OutPut of: 950524AI Operator :DDW Date of the Analysis 1995-06-24 07:50 Analysis FileName C:kSKALARXDATA\HWIDATA\LIVERSkgSO524AI HWll59L2.XLT 1 Tracer 2 Ddft 3 Wash 4 Standard 1 5 Standard 2 6 Standard 3 7 Standard 4 8 Standard 5 9 Standard 6 10 Standard 7 11 Standard 8 12 Standard 9 13 Standard 10 14 Ddft 15 Wash 16 F54092-1 17 F54092-2 18 F54092-3 19 F54106-1 20 F54106-2 21 F54106-3 22 BLK 23 SPK 1 24 SPK 2 25 SPK 3 26 Drift 27 Wash -C) 28 SPK 4 29 SPK 5 30 SPK 6 1.50 1.50 0.015 0.03 0.06 0.09 0.12 0.15 0.30 0.60 1.20 1.50 1.50 1.50 1.45 97% 1.47 98% 0.00 0.02 107% 0.03 94% 0.06 101% 0.09 103% 0.12 97% 0.16 103% 0.28 94% 0.61 102% 1.24 103% 1.46 97% 1.50 100% 0.00 0.06 0.05 0.04 0.06 0.06 0.04 0.01 0.08 0.08 0.09 1.45 97% 0.00 0.15 0.13 0.14 2.0 0.1169 0.96 78.36 75.61 2.0 0.1453 0.69 78.36 54.15 2.0 0.1381 0.60 78.36 46.75 2.0 0.1336 0.85 82.301 70.35 2.0 0.1249 0.90 82.301 74.33 2.0 0.1213 0.71 82.301 58.76 2.0 0.1326 0.18 2.0 0.1133 1.34 0.004 63.00 2.0 0.1577 0.99 0.004 63.00 2.0 0.1441 1.19 0.004 63.00 2.0 0.1556 1.89 2.0 0.1474 1.72 2.0 0.1518 1.82 Page 1 0.004 0.004 0.004 126.00 126,00 126.00 HWI 159L2.XLT 31 LIVER BLK-1 0.05 32 LIVER BLK-2 0.03 33 LIVER BLK-3 0.02 34 SPK 63-1 0.09 35 SPK 63-2 0.09 36 SPK 63-3 0.15 37 SPK 63-4 0.09 36 Drift 1.50 1.49 100% 39 Wash 0.00 40 F54088-1 0.05 41 F54088-2 0.05 42 F54088-3 0.04 43 BLK 4 0.02 44 SPK 63-1 0.12 45 SPK 63-2 0.08 46 SPK 63-3 0.08 47 SPK 63-4 0.08 48 BLK-1 0.02 49 BLK-2 50 Drift 0.00 1.50 1.49 99% 51 Wash 0.00 52 SPK-1 0.08 53 SPK-2 0.10 54 BLK 3 0.02 55 SPK-3 0.10 56 SPK-4 0.02 57 F54089-1 0.03 58 F54089-2 0.03 59 F54089-3 0.03 60 F54099-1 0.04 61 F54099-2 0.04 62 Drift 1.50 1.49 99% 63 Wash 0.00 64 F54099-3 0.05 65 F54114-1 0.03 66 F54114-2 0.03 2.0 0.1467 0.70 2.0 0.1328 0.52 2.0 0.1101 0.29 2.0 0.1425 1.25 2.0 0.1316 1.36 2.0 0,1426 2.03 2.0 0.1079 1.73 0.004 0.004 0.004 0.004 63.00 63.00 63.00 63.00 2.0 0.1448 0.72 84.26 60.63 2.0 0.1228 0.89 84.26 75.07 2.0 0.1317 0.63 84.26 53.49 2.0 0.1227 0.35 2.0 0.1358 1.71 2.0 0.1393 1.22 2.0 0.1468 1.15 2.0 0.1494 1.14 0.004 0.004 0.004 0.004 63.00 63.00 63.00 63.00 2.0 0.1643 0.24 2.0 0.1578 0.06 2.0 0.1283 1.32 2.0 0.1292 1.49 0.004 0.004 63.00 63.00 2.0 0.1396 0.24 2.0 0.1325 1.48 2,0 0.1109 0.30 0.004 0.004 63.00 63.00 2.0 0.1532 0.42 71.796 29.81 2.0 0.1568 0.41 71.796 29.12 2.0 0.1474 0.39 71.796 28.15 2.0 0.1386 0.56 82.143 46.35 2.0 0.1403 0.53 82.143 43.21 2.0 0.1575 0.59 82.143 48.29 2.0 0.1573 0.39 80.966 31.19 2.0 0.1359 0.38 80.966 31.10 Page 2 HWll59L2.XLT 67 F54114-3 0.03 68 F541 09-1 0.03 69 F54109-2 0.03 70 F54109-3 0.03 71 F54080-1 0.03 72 F54080-2 0.03 73 F54080-3 0.00 74 Drift 1.50 1.47 98% 75 Wash 0.00 2.0 0.1552 0.40 80.966 32.14 2.0 0.1326 0.38 73.958 28.45 2.0 0.1339 0.38 73.958 28.17 2.0 0.1536 0.45 73.958 33.13 2.0 0.1322 0.46 75.42 34.91 2.0 0,1323 0.50 75.42 37.74 2.0 0.1028 0.00 75.42 0.00 Page 3 cn 995-05-23 15:26 output of 950523Al .oftware version 6.1 cl990,93 perator : DDW ate of the Analysis : 1995-05-23 10:18 .nalysis File Name : C:\SKALA-R\DATA\HWIDATA\LIVERS\950523Al Z7- Aokb-rqlocis. I (,*-3Z9 -1S'oiI.;,@ 'luoride 1.5 'alibration order = Inverse Logarithm :.lope s x - cl lesult = 101 s 1 x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode 12 = il = Lo = -0.00000 0.00065 -1.22333 .'luoride L .1 .alibration order 2 7or.relation r = 0 .99847 "esult = a2 X2 + al * x + aO t2 = il = 10 = -0.00000 0.00024 -0.00181 @ampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SA1000 :1 : 50 sec. : 120 sec. : I sec. : Single : None : 70 mm. )iluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample :1 Dilution runs : I User file : TXT Reproces : No 0()00!)!) ir -995-05-23 15:26 OutPut of : 950523Al 7luoride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 Debubbled Yes 0 No No : 4095 :0 : Off si standard : Ignore s2 standard : Ignore s3 standard : Ignore s4 standard : Ignore s5 standard : Ignore s6 standard : 0.150 s7 standard : 0.300 S8 standard : 0.600 S9 standard : 1.200 slO standard : 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No form.Ula output Tluoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event :0 : Debubbled : No :0 : No : No : 4095 :0 : Off o(loloo .995-05-23 15:26 OutPut of sl standard : 0.015 s2 standard : 0.030 s3 standard : 0.060 s4 standard : 0.090 s5 standard : 0.120 s6 standard : 0.150 s7 standard : Ignore s8 standard : Ignore S9 standard : Ignore sio standard : Ignore order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape. : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 Filter No Regeneration No formula c4:=c3 output 950523Al ooolol 995-05-23 15:26 OutPut of : 950523Al Fluoride 1.5 Fluoride L PPM PPM os Typ Ident Ch Result F Time Ch Result F Time iw t d w sl s2 s3 s4 s5 s6 .0 s7 - S8 2 s9 3 slO 4d 5w 6u 7u 3u 9u 0u 1u 2u 3u 4 1-1 5u .6 d .7 w .3 u 9u 0u Iu 2u @3 u 4u u 6u @7 u 8d 9w -0 u :1 u -2 u -3 u -4 u -5 u 6u -7 u 3u .9 u -.0 d @l w u Initial Wash Tracer Drift Wash Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Standard 8 Standard 9 Standard 10 Drift Wash BLK 1 BLK 2 BLK 3 F54075-1 F54075-2 F54075-3 P54081-1 F54081-2 F54081-3 F54105-1 Drift Wash F54105-2 F54105-3 F54107-1 F54107-2 F54107-3 F54074-1 F54074-2 F54074-3 F54085-1 F54085-2 Drift Wash F54085-3 BLK 1 BLK 2 SPK 1 SPK 2 SPK 3 SPK 4 SPK 5 SPK 6 BLK 1 Drift Wash BLK 2 SPK 1 3 0.060 3 1.472 3 1.476 3 0.060 3 0.068 3 0.071 3 0.088 3 0.108 3 0.127 3 0.154 3 0.285 3 0.611 3 1.238 3 1.461 3 1.532 3 0.060 3 0.084 3 0.076 3 0.077 3 0.074 3 0.075 3 0.069 3 0.069 3 0.069 3 0.067 3 0.068 3 1.526 3 0.060 3 0.070 3 0.070 3 0.866 3 0.083 3 0.072 3 0.070 3 0.068 3 0.070 'a 0.068 3 0.070 3 1.515 3 0.060 3 0.087 3 0.071 3 0.072 '1 0.151 3 0.112 3 0.119 3 0.104 3 0.130 3 0.106 Z' 0.080 3 1.533 3 0.060 3 0.069 3 0.119 65 213 389 627 740 916 1090 1264 1442 1616 1792 1966 2140 2314 2490 2723 2839 3014 3191 3366 3541 3717 3891 4065 4238 4415 4591 4832 4941 5115 5291 5465 5640 5820 5992 6166 6342 6514 66 92 6925 7044 7216 7393 7583 7744 7918 8095 8271 8445 3619 8793 9030 9141 9319 4 0 4 0.5569 0 4 0.5576 0 4 #.#### 0 4 0.0188 0 4 0.0255 0 4 0.0599 0 4 0.0917 0 4 0.1189 0 4 0.1501 0 4 0.2516 0 4 0.3836 0 4 0.5188 0 4 0.5552 0 4 0.5664 0 4 #.#### 0 4 0.0527 0 4 0.0354 .0 4 0.0393 0 4 0.0327 0 4 0.0331 0 4 0.0207 0 4 0.0200 0 4 0.0211 0 4 0.0169 0 4 0.0188 0 4 0.5654 0 4 #.#### 0 4 0.0241 0 4 0.0220 0 4 0.4478 V%x "5&- 4 0.0507 0 4 0.0274 0 4 0.0230 0 4 0.0183 0 4 0.0220 0 4 0.0174 0 4 0.0220 0 4 0.5638 0 4 #.#### 0 4 0.0586 0 4 0.0244* 0 4 0.0285 0 4 0.1464 0 4 0.0984 0 4 0.1080 0 4 0.0860 0 4 0.1228 0 4 0.0901 0 4 0.0446 0 4 0.5666 0 4 0 4 0.0218 0 4 0.1076 0 Page 1 of 2 0 0 10 995-05-23 15:26 output of 950523AI Fluoride 1.5 Fluoride L PPI%F PPM os Typ Ident Ch Result F Time Ch Result F Time 4u SPK 2 3 0.114 9495 4 0.1015 0 3u SPK 3 3 0.110 9670 4 0.0956 0 6u SPK 4 3 0.118 9845 4 0.1065 0 7u F54082-1 3 0.124 10020 4 0.1151 0 3u F54082-/@. 3 0.128 10196 4 0.1204 0 9u F54082-3 3 0.124 10370 4 0.1142 0 0u F54 111-1 3 0.103 10546 4 0.0953 0 1u F54111-2 3 0.098 10720 4 0.0771 0 2d Drift 3 1.512 10394 4 0.5633 0 3w Wash 3 0.060 11133 4 #.#### 0 4u F54111-3 3 0.114 11245 4 0.1017 0 1u F54079-1 3 0.124 11420 4 0.1149 0 6u F54079-2 3 0.119 11595 4 0.1082 0 7u F54079-3 3 0.115 11770 4 0.1024 0 3u F54116-1 3 0.138 11944 4 0.1315 0 9u F54116-2 3 0.127 12118 4 0.1181 0 0u F54116-3 3 0.138 12296 4 0.1317 0 1u F54112-1 3 0.130 12470 4 0.1219 0 '2 F54112-2 3 0.150 12645 4 0.1460 0 3u F54112-3 3 0.137 12821 4 0.1306 0 4d Drift 3 1.483 12995 4 0.5587 0 3w Wash 3 0.060 13234 4 #.#### 0 6u F54076-1 3 0.154 13347 4 0.1501 0 7u F54076-2 3 0.151 13520 4 0.1462 0 3u F54076-3 3 0.139 13697 4 0.1336 0 9u F54113-1 3 0.128 13873 4 0.1196 0 0u F54113-2 3 0.123 14047 4 0.1134 0 1u F54113-3 3 @0.123 14221 4 0.1134 0 2u F54084-1 3 0.145 14397 4 0.1399 0 3u F54084-2 3 0.157 14571 4 0.1530 0 4u F54084-3 3 0.139 14747 4 0.1330 5u F54098-1 3 0.136 14921 4 0.1296 0 6d Drift 3 1.490 15095 4 0.5593 0 7w Wash 3 0.060 15327 4 #.#### 0 3u F54098-2 3 0.143 15447 4 0.1380 0 u F54098-3 3 0.139 15621 4 0.1327 0 u F54087-1 3 0.077 15796 4 0.0384 0 1u F54037-2 3 0.073 15969 4 0.0308 0 2u F54087-3 3 0.075 16142 4 0.0343 0 u F54088-1 3 0.1-23 16322 4 0.1140 0 u F54088-2 3 0.082 16496 4 0.0484 0 5u F544*038-'o 3 0.081 16672 4 0.0457 0 bd Dri-ft 'D 1.489 16846 4 0.5596 0 7w Wash 3 0.061) 17079 4 0 rw Runout Wash 3 0.060 1731el 4 0 Page 2 of 2 .......... ........... ....................... r ::i ............. ....... . .............. .......... .......................... ....... ............ .............. ............ ............ ..................... ................. --------- .. .............. .... ... ............ ........ Lr: .......... .......... ............. . ................................................. ........................................... .......... - -------.-.-.-.-.-..-...--.-.-..-..-........................................................ .. ..................................... ............. . CL ............. ..................................... ............ ql .. ....... .............................................. .................................. .. ............ ............... . .................................... ... ................................... ....................... ................ ... . ......-. -------- . ......... .......... .......................... Tip en ............ 7 - ------------- . ................ .......... ............ .. .......... . ................ ........... ..... . . .............. ....-.-. ---- ............. - ----- . ............................. ------------- .............. .......... ........... ----------- cr) fl,l ......... . .......... .....................-.-.----------- Li.4 C-T- ---------------- l',041 ..... ............ .............. ..................... ......... ........... . ...... . ...... .............. .... ............ .......... . .......... ........................... ............... ................................... .............. ................ .............. .......... cl I ......................... ----------,- * .............. . .......... ................ ....................... .......... ............ .......... .......................................................................... ............. . ................................. .............. ........... lr ........... . ...... 995-05-24 12:48 Output of 950524AI :iersion 6.1 cl990,93 ..,oerator DDW ate of the An-alysis 1995-05-24 07:50 ..nalysis File Name C:\SKALAR\DATA\HWIDATA\L'LVERS\950524A! C*AA> 140\D-T 'luoride 1.5 :alibration order = Inverse Logarithm )'olp e s r X - cl @.esult = 10L s x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode -2 = -il= .10 = -0.00000 0.00065 -1.22861 .7luoride L :alibration order = 2 7o.rx-elation r = 0. 99892 -lesult = a2 X2 + al * x + aO .a2 = @l = 10 = 0.00000 0.00021 -0.00134 3a,T,pler Type Number Sample Time Wash Time Air Time Take up special needle Height SA1000 :1 : 50 sec. : 120 sec. I sec. Single None 70 mm. -,i-.I.uter needle Height dilution Factor dilution Volume Resample Dilution runs 80 mm 10 2.5 ml. 1 1 Use4- file : TXT Reproces : No c-l5 IL .995-05-24 12:48 output of 950524Al 7luoride 1.5 Path number Signal type Decolor syste-,,nNumber dilute Resanir)le dil Threshold diG output Window event 3 Debubbled Yes :0 : No : No : 4095 :0 : Off sl standard : Ignore s2 standard : Ignore s3 standard : Ignore s4 standard : Ignore s5 standard : Ignore s6 standard : 0.150 s7 standard : S8 standard : 0.300 0.600 S9 standard : 1.200 slO standard : 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1900 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula outnut 71-tiorideL Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : b-;-:f -995-05-24 12:48 OutPut of 950524Al si standard 0.015 S2 standard 0.030 s3 standard 0.060 s4 standard 0.090 s5 standard 0.1'ZIO s6 s,raniard 0.150 s7 standard Ignore ss standard Ignore S9 star-dard Ignore slO standard Ignore order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formU'La c4:=c3 our-put .995-05-24 12:48 output of 1)50524AI Fluoride 1.5 Fluoride L ppm PPM .,os Typ Ident Ch Result F Time Ch Result F Time it iw jrit-Lal Wash 3 0,059 65 4 0 t Tracer 3 1.452 211 4 0.8505 0 d Drift 3 1.469 387 4 0.8584 0 w Wash 3 0.059 626 4 #.#### 0 si Standard 1 3 0.067 7'36 4 0.0160 0 s2 standard 2 3 0.073 912 4 0.0282 0 s3 standard 3 3 0.090 1086 4 0.0603 0 s4 Standard 4 'a 0.110 1262 4 0.0931 0 s5 standard 5 3 0.127 1438 4 0.1164 0 4 s6 Standard 6 3 0.155 1612 4 0.1510 0 .0 s7 standard 7 3 0.283 1788 4 0.2712 0 -J. ss Standard 8 3 0-614 1964 4 0.4713 0 '.2 s9 Standard 9 3 1.237 2138 4 0.7522 0 3 sio Standard 10 3 1.462 2312 4 0.8550 0 .4 d Drift 3 1.495* 2488 4 0.8710 0 -5 w Wash 3 0.059 2725 4 #.#### 0 u F54092-1 3 0.088 2838 4 0.0564 0 -7 u F54092-2 3 0.084 3014 4 0.0502 0 -8 u F54092-3 3 0.079 3190 4 0.0412 0 -9 u F54106-1 3 0.088 3367 4 0.0571 0 -,0 u F54106-2 3 0.088 3541 4 0.0564 0 u F54106-3 3 0.080 3713 4 0.0433 0 ,2 u BLK 3 0.065 3883 4 0.0118 0 '@3 u SPK 1 3 0.099 4063 4 0.0760 0 -@4 u SPK 2 3 0.101 4241 4 0.0784 0 25 u SPK 3 3 0.105 4415 4 0.0854 0 '6 d Drift 3 1.454 4589 4 0.8511 0 L7 w Wash 3 0.059 4830 4 0 SPK 4 3 0.152 4939 4 0.1473 0 '9 u SPK 5 3 0.135 5113 4 0.1269 0 :0 u SPK 6 3 0.144 5291 4 0.1380 0 u LIVER BLK-1 3 0.085 5463 4 0.0515 0 u LIVER BLK-2 3 0.076 5639 4 0.0346 0 33 u LIVER BLK-3 3 0-067 5813 4 0.0162 0 '@-4 u SPK 63-1 3 0.108 5992 4 0.0889 0 5u SPK 63-2 3 0.108 6168 4 0.0898 0 u SPK 63-3 3 0.150 6342 4 0.1450 0 u SPK 63-4 3 0.110 6516 4 0.0931 0 d Drift 3 1.494 6692 4 0.8707 0 39 w Wash 3 0.059 6923 4 0 @O u F54o88-l 3 o.085 7040 4 0.0521' 0 u F54088-2 3 0-087 7218 4 0.0547 0 @12 u F54088-3 3 0.080 7390 4 0.0418 0 -1:3 u BLK 4 3 0.069 7566 4 0.0217 0 -!4 u sPK 63-1 3 0.127 7742 4 0.1162 0 '5 u SPK 63-0- 3 0.105 7916 4 0.0847 0 46 u SPK 63-3 @a 0.105 8092 4 0.0847' 0 u SPK 63-4 3 0.105 8268 4 0.0849 0 u BLK-1 3 0.069 8442 4 0.0198 0 -r9 u BLK -2 3 0.062 8614 4 0.0049 0 50 d Drift 1.487 6791 4 0.8670 0 ;l W Wash 0.059 9029 4 #.#### 0 =j2 u SPK-1 3 0.105 9143 4 0.0847 0 u SPK-2 0-112 9317 4 0.0960 0- Pa ge I of 2 oooll, 995-05-24 12:48 @output of 950524AI Fluoride 1.5 Fluoride L PPH PPM os Typ Ident Ch Result F Time Ch Result F Time 4u BLK 3 3 0.067 9491 4 0.0166 0 5u SPK-3 3 0.114 9669 4 0.0979 0 6u SPK-4 3 0.067 9839 4 0.0166 0 7u F54089-1 3 0.074 10015 4 0.0318 0 8u F54089-'L' 3 0.074 10193 4 0.0318 0 9u F54089-3 3 0.073 1-0368 4 0.0289 0 0u F54099-1 3 0.078 10544 4 0.0391 0 1u F54099-2 3 0.077 10718 4 0.0369 0 2d Drift 3 1.487 10893 4 0.8670 0 .3 w Wash 3 0.059 11107 4 #.#### 0 )4 u F54099-3 3 0.082 11244 4 0.0463 0 .i u F54114-1 3 0.074 11416 4 0.0303 0 6u F54114-2 3 0.072 11594 4 0.0261 0 7u F54114-3 3 0.074 1176-8 4 0.0308 0 8u F54109-1 3 0.071 11940 4 0.0255 0 9u F54109-2 3 0.071 12115 4 0.0255 0 0u F54109-3 3 0.076 12296 4 0.0344 0 1u F54080-1 3 0.074 12469 4 0.0306 0 2u F54080-2 3 0.075 12645 4 0.03.31 0 3u F54080-3 3 0.059 A 12818 4 0 4d Drift 3 1.468 12993 4 0.8578 0 5w Wash 3 0.059 13185 4 0 't rw Runout Wash 3 0.059 13468 4 0 Page 2 of 2 oooll,l irTTO()O p ':-TAe -1 ........ . ....... .............. 4-1 LI-iir.*'--9-1- i.-a . ................................. Lei iT, i,ri ................. ............. . ....... ..... .......................................... . ........... elWl% ..JO4 ..................... ...................... ... ...... .............................................. . . ......... - - ------------ f::rI ..... ........... . ....... ........ . .......... . ...... . ........ .................. .......... . ......... . ...................... ... ...... .. ................. . .............. ........ - --------- .... . ..... og .......... . ....... .................... ......------ ----- ............ ..................... .... ......... ....... ....................... ...... ----------------------- ............ -----.-....... ................ ................................... . ............... ............... . .......... .. ........... .................... .......... if.............. . ......-.-.----- LA ... .............. ............ ............ .............. .......... . ............ Fl.un-ridie.- lip c: 9.11.3Summary and raw data;ppm F- in serum as determinedby thermalextractiofnollowedby analysisusing Orion ion analyzer. 000120 HWI 6329-159 AMDT 042095.1 Dohrmann Serum Analysis AnalysisDates: 04/28/95- 05110195 All serum sampleswere thermallyextractebdy a modifiedDohrmann DX2000 OrganicHalideAnalyzerand collecteidna 1:1 milliQ water and TISAB solution.Ile sampleswere measured on an Orion EA940 expandableion analyzer.The Dohrmann was calibrateudsing32ppm, 63ppm, 126ppm, and 253ppm FC-95 standards.The Orion was calibrated by directmeasurement with no blankcorrectiounsing0.05ppm, 0.Ippm, 0.5ppm, l.Oppm and 1.5ppm F-standards.T'heslope,intercepta,nd correlatiownere recordedintheappropriatleogbook. A summary tableisincluded,showing theppm F-ineach sample (seepage 2). The valueof'74D"has been enteredforany sample withan Orion readingof below 0.05. Pages 3 - 10 show theexcelspreadsheethatwas generatedwhen the samples were analyzed.Pages 11 - 18 show thesame spreadsheetwith "ND" insertedwhere theOrion readingisbelow 0.05. Page I of 18 FC-95 PK Dosage: 0 rng/kg HM 6329-159 Fluodde concentrafioninrabbkserum (ppm F-) Sample 4 hour 8 hour 12 hour 24 hour 48 hour F54074 ND ND ND ND F54075 NO ND ND ND ND F54081 ND ND ND ND F54085 ND ND ND ND F54086 ND ND ND ND F54104 NO ND ND ND F54105 ND ND ND ND ND F54107 ND ND NO ND Dosage: 5 rng/kg Sample F54077 F54083 F54091 F54094 F54097 F541 00 F54102 F54108 4 hour ND NO ND ND ND ND ND N0 8 hour ND ND NO ND ND ND ND ND 12 hour 24 hour ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Dosage: 10 mg/kg Sample F54071 F54078 F54090 F54093 F54095 F54096 F54101 F541 10 4 h2ur ND ND NO ND ND NO ND ND 8 hour ND NO ND ND ND ND ND 1.5 12 hour 24 hour ND ND ND ND ND ND ND ND NO ND ND ND NO ND ND ND Dosage: 100 mg/kg Sarnple F54080 F54087 F54088 F54089 F54092 F54099 F54106 F54109 F54114 4 hour ND ND ND ND ND ND ND ND ND 8 hour ND ND ND ND ND ND NO ND ND 12 hour 15 hour 24 hour 48 hours ND ND ND ND ND ND ND ND ND ND 1.5 ND NO ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1.7 ND ND ND ND ND ND Day 8 ND ND ND ND ND ND ND ND ND Day 22 Day 28 ND ND ND ND ND ND ND ND ND Dosage: 500 mg/kg Sample F54076 F54079 F54082 F54084 F54098 F541 11 F54112 F54113 F54116 4 hour ND 2.3 1.1 1.3 ND 1.9 ND 1,6 2.0 8 hour ND ND 1.3 ND 1.0 ND 1.2 ND 3.4 12 hour ND ND NO ND ND ND 1.3 ND ND 24 hour 48 hours Day 8 360 hour 1.2 1.2 1.4 1.3 1.1 1.3 1.5 ND 1.2 1.8 1.5 1.4 1.3 1.3 1.3 1.1 1.3 1.5 1.3 1.3 1.0 1.3 1.2 ND 1.3 1.4 1.6 1.3 1.3 1.7 1.3 1.4 Day 22 ND ND ND ND Day 28 ND ND ND ND 6329159S-XLS STUDY 8329-159 . ....... ... ......... serum blank-1 serum blank-2 serum spike 63-1 serum spike 63-2 serum spike 94.5-1 serum spike94.5-2 serum spike126-1 serum spike126-2 serum spike 126-3 serum spike 126-4 F54081-4 F54075-4 F54107-4 F54105-4 F54074-4 Blank1 Blank 2 Blank3 Blank4 Blank5 Slar* 6 Serum Spk-1 Serum Spk-2 Serum Spk-3 Serum Spk-4 Serum Spk-5 Serum Spk-B Serum Spk-7 Serum Spk-B Sarum Spk-9 Serum Spk-10 FW85-4 FW86-4 F54104-4 FW77-4 FW83-4 F54094-4 F54100-4 FW91-4 serumblk1 serum bik 2 serum spk 1 serumspk 2 F54097-4 F54102-4 F54108-4 F54071-4 u 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 u on**,@:@:rienag.:....M....... 63 63 94.5 94.5 126 126 126 126 63 63 63 126 126 126 126 126 126 126 63 63 0.036 0.020 0.070 0.070 0.063 0.046 0.15 0.099 0.15 0.13 0.040 0.032 0.024 0.025 0.034 0.71 0.34 0.36 0.084 0.038 0.039 0.055 0.068 0.073 0.10 0.11 0.11 0.11 0.098 0.12 0.10 0.045 0.027 0.025 0.021 0.020 0.021 0.020 0.019 0.029 0.024 0.071 0.072 0.022 0.020 0.016 0.017 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10. 0.10 0.10 0.10 . .-ISA a ....... -F - L-,*---6-A-i-W------ 2.0 0.72 2.0 0.40 2.0 93% 1.4 2.0 92% 1.4 2.0 56% 1.3 2.0 41% 0.92 2.0 101% 3.1 2.0 65% 2.0 2.0 98% 3.0 2.0 85% 2.6 2.0 0.80 2.0 0.65 2.0 0.48 2.0 0.51 2.0 0.67 2.0 14 2.0 6.9 2.0 7.2 2.0 1.7 2.0 0.75 2.0 0.79 2.0 73% 1.1 2.0 90% 1.4 2.0 96% 1.5 2.0 67% 2.0 2.0 74% 2.2 2.0 73% 2.2 2.0 72% 2.2 2.0 65% 2.0 2.0 77% 2.3 2.0 68% 2.1 2.0 0.90 2.0 0.54 2.0 0.49 2.0 0.42 2.0 0.40 2.0 0.42 2.0 0.41 2.0 0.38 2.0 0.59 2.0 0.48 2.0 94% 2.0 95% 2.0 1.4 1.4 0.45 2.0 0.41 2.0 0.32 2.0 0.34 ........ -F 0.15 0.15 0.23 0.23 0.30 0.30 0.30 0.30 0.15 0.15 0.15 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.15 0.15 ....d... 0.072 0.040 0.14 0.14 0.13 0.092 0.31. 0.20 0.30 0.26 0.080 0.065 0.048 0.051 0.067 1.4 0.69 0.72 0.17 0.075 0.079 0.11 0.14 0.15 0.20 0.22 0.22 0.22 0.20 0.23 0.21 0.090 0.054 0.049 0.042 0.040 0.042 0.041 0.038 0.059 0.048 0.14 0.14 0.045 0.041 0.032 0.034 6329159S.XLS STUDY 6329-159 . ....... ............. FW93-4 FW95-4 F54101-4 F54078-4 F54090-4 FW96-4 F54110-4 F54087-4 F54088-4 F54089-4 F54106-4 FW80-4 F54092-4 F54109-4 F54114-4 FW76-4 F54082-4 F54111-4 F54113-4 FW79-4 F54084-4 F54098-4 F54116-4 FW75-8 FWS 1-8 F54105-8 F54107-8 FW74-8 FWSS-8 F54086-8 serum bik 1 serum bik 2 serum spk 1 serum spk 2 F54104-8 FW77-8 F54083-8 FW94-8 F54100-8 F5409 1-8 FW97- 8 F54102- 8 F$4108- 8 F54071 - a F54093 -8 FMSS -8 FS4101- 8 F54078-8 @.is --k-d 0.004 0.004 .............. ... .................... MI n:T..e m 'a -.p..p.m.;........ ------ 0.026 -10 2.0 0.016 0.10 2.0 0.015 0.10 2.0 0.021 0.10 2.0 0.020 0.10 2.0 0.017 0.10 2.0 0.020 0.10 2.0 0.025 0.10 2.0 0.028 0.10 2.0 0.020 0.10 2.0 0.032 0.10 2.0 0.025 0.10 2.0 0.019 0.10 2.0 0.021 0.10 2.0 0.039 0.10 2.0 0.041 0.10 2.0 0.057 0.10 2.0 0.096 0.10 2.0 0.082 0.10 2.0 0.12 0.10 2.0 0.067 0.10 2.0 0.046 0.10 2.0 0.10 0.10 2.0 0.025 0.10 2.0 0.020 0.10 2.0 0.021 0.10 2.0 0.015 0.10 2.0 0.015 0.10 2.0 0.021 0.10 2.0 0.031 0.10 2.0 0.029 0.10 2.0 0.028 0.10 2.0 63 0.080 0.10 2.0 63 0.069 0.10 2.0 0.031 0.10 2.0 0.023 0.10 2.0 0.018 0.10 2.0 0.025 0.10 2.0 0.017 0.10 2.0 0.014 0.10 2.0 0.014 0.10 2.0 0.013 0.10 2.0 0.013 0.10 2.0 0.015 0.10 2.0 0.019 0.10 2.0 0.026 0.10 2.0 0.016 0.10 2.0 0.021 0.10 2.0 .. L ............................ . ......... ::rcw u 0.4-1 ---------------- 0.32 0.032 0.29 0.029 0.41 0.041 0.39 0.039 0.34 0.034 0.39 0.039. 0.50 0.050 0.55 0.055 0.41 0.041 0.63 0.063 0.51 0.051 0.38 0.038 0.42 0.042 0.78 0.078 0.81 0.081 1.1 0.11 1.9 0.19 1.6 0.16 2.3 0.23 1.3 0.13 0.93 0.093 2.0 0.20 0.49 0.049 0.40 0.040 0.43 0.043 0.30 0.030 0.30 0.030 0.43 0.043 0.62 0.062 0.58 0.058 0.56 0.056' 106% 1.6 0.15 0.16 91% 1.4 0.15 0.14 0.62 0.062 0.46 0.046 0.35 0.035 0.51 0.051 0.35 0.035 0.28 0.028 0.28 0.028 0.26 0.026 0.25 0.025 0.30 0.030 0.37 0.037 0.51 0.051 0.32 0.032 0.42 0.042 J4 63291 59S.)(LS ............ FW90- 8 F54096- 8 F541 10- 8 FW87- 8 F54088-8 F54089-8 F54080-8 F54106- 8 FM92- 8 F$4109- 8 F54114- 8 FM76- 8 F54082-8 F541 11- 8 F54113- 8 F$4079-8 FW84- 8 F54098-8 F54116- 8 serum bik 1 serum bik2 serum spk 1 serum spk 2 F54075-12 FW81-12 F54105-12 F54107-12 FM74-12 F$4085-12 FW88- 12 F54086-12 FM77-12 F54083-12 F54094-12 F54100-12 F54091-12 FW97-12 F64102-12 F54108-12 FW77-12 F54071-12 F54093-12 FWSS-L 2 F54101-12 F54110-12 F54096-12 F54090-12 F54078-12 . .. ....... ua:.:.:.:.....U;..... .. ........-.C... ........itd."Or*" S.SMP .uo.*:*:u,stoon::.-.:..........M.L..... .1n0 0.02i- ----0 ..-..h.a.m........... :i .0 0.017 0.10 2.0 0.075 0.10 2.0 0.022 0.10 2.0 0.033 0.10 2.0 0.031 0.10 2.0 0.032 0.10 2.0 0.043 0.10 2.0 0.037 0.10 2.0 0.024 0.10 2.0 0.034 0.10 2.0 0.045 0.10 2.0 0.063 0.10 2.0 0.044 0.10 2.0 0.048 0.10 2.0 0.042 0.10 2.0 0.045 0.10 2.0 0.050 0.10 2.0 0.17 0.10 2.0 0.012 0.10 2.0 0.018 0..10 2.0 0.004 63 0.066 0.10 2.0 87% 0.004 63 0.070 0.10 2.0 93% 0.028 0.10 2.0 0.021 0.10 2.0 0.026 0.10 2.0 0.019 0.10 2.0 0.017 0.10 2.0 0.018 0.10 2.0 0.020 0.10 2.0 0.025 0.10 2.0 0.038 0.10 2.0 0.032 0.10 2.0 0.035 0.10 2.0 0.022 0.10 2.0 0.029 0.10 2.0 0.025 0.10 2.0 0.022 0.10 2.0 0.014 0.10 2.0 0.046 0.10 2.0 0.021 0.10 2.0 0.016 0.10 2.0 0.016 0.10 2.0 0.020 0.10 2.0 0.016 0.10 2.0 0.012 0.10 2.0 0.017 0.10 2.0 0.014 0.10 2.0 pp 0.42 0.34 1.5 0.44 0.66 0.62 0.64 0.86 0.74 0.49 0.68 0.89 1.3 0.88 0.95 0.84 0.89 1.0 3.4 0.25 0.36 1.3 1.4 0.56 0.42 0.53 0.39 0.35 0.37 0.41 0.50 0.76 0.64 0.71 0.44 0.58 0.49 0.44 0.29 0.92 0.41 0.33 0.32 0.40 0.31 0.25 0.34 0.28 STUDY# 6329-159 "-k 0.15 0.15 U 0.042 0.034 0.15 0.044 0.066 0.062 0.064 0.086 0.074 0.049 0.068 0.089 0.13 0.088 0.10 0.084 0.089 0.10 0.34 0.025 0.036 0.13 0.14 0.056 0.042 0.053 0.039 0,035 0.037 0.041 0.050 0.076 0.064 0.071 0.044 0.058 0.049 0.044 0.029 0.092 0.041 0.033 0.032 0.040 0.031 0.025 0.034 0.028 01 6329159S.XLS F54088-12 FS4106-12 F54089-12 F54087-12 FW80-12 F54092-12 F54109-12 F54114-12 F54076-12 F54113-12 FW82-12 F54111-12 FW98-12 F54084-12 F54079-12 F54116-12 Serum ENk-1 Serum Bik-2 Serum Spk-1 Serum Spk-2 Serum Spk-3 Serum Spk-4 Serum Spk-5 Serum Spk-6 Serum Spk-7 Serum Spk-B F54105-24 F54075-24 FW81-24 F54107-24 F54104-24 F54086-24 FW74-24 Blank 1 Blank 2 spike 1 spike 2 spike 3 spike 4 spike 5 spike 6 Blank 3 Blank 4 FWSr),-24 F54083-24 F54100-24 F54077-24 FW94-24 F54091-24 . ........................... 60 ............. ......... ... .......A..... ........... 0.025 0.10 2.0 0.027 0.10 2.0 0.019 0.10 2.0 0.019 0.10 2.0 0.018 0.10 2.0 0.017 0.10 2.0 0.021 0.10 2.0 0.026 0.10 2.0 0.027 0.10 2.0 0.022 0.10 2.0 0.034 0.10 2.0 0.046 0.10 2.0 0.042 0.10 2.0 0.044 0.10 2.0 0.043 0.10 2.0 0.047 0.10 2.0 0.033 0.10 2.0 0.030 0.10 2.0 0.004 63 0.004 63 0.004 63 0.004 63 0.034 0.040 0.050 0.054 0.10 0.10 0.10 0.10 2.0 44% 2.0 52% 2.0 66% 2.0 72% 0.004 63 0.083 0.10 2.0 110% 0.004 63 0.053 0.10 2.0 71% 0.004 63 0.049 0.10 2.0 65% 0.004 63 0.055 0.10 2.0 73% 0.041 0.10 2.0 0.022 0.10 2.0 0.017 0.10 2.0 0.015 0.10 2.0 0.018 0.10 2.0 0.016 0.10 2.0 0.013 0.10 2.0 0.031 0.10 2.0 0.022 0.10 2.0 0.004 63 0.004 63 0.004 63 0.033 0.044 0.047 0.10 0.10 0.10 2.0 43% 2.0 58% 2.0 63% 0.004 63 0.047 0.10 2.0 62% 0.004 63 0.052 0.10 2.0 68% 0.004 63 0.051 0.10 2.0 67% 0.036 0.10 2.0 0.020 0.10 2.0 0.021 0.10 2.0 0.019 0.10 2.0 0.016 0.10 2.0 0.017 0.10 2.0 0.015 0.10 2.0 0.016 0.10 2.0 0.51 0.54 0.38 0.37 0.36 0.33 0.41 0.52 0.55 0.44 0.69 0.91 0.84 0.87 0.85 0.94 0.66 0.60 0.67 0.79 0.99 1.1 1.7 1.1 0.99 1.1 0.81 0.44 0.35 0.30 0.37 0.33 0.27 0.61 0.43 0.65 0.88 0.95 0.94 1.0 1.0 0.72 0.39 0.41 0.38 0.32 0.33 0.31 0.31 STUDY# 6329-159 ............. ma k. ........ ...6..d.................... 0.051 0 054 0.'038 0.037 0.036 0.033 0.041. 0.052 0.055 0.044 0.069 0.091 0.084 0.087 0.085 0.094 0.066 0.060 0.15 0.067 0.15 0.079 0.15 0.10 0.15 0.11 0.15 0.17 0.15 0.11 0.15 0.10 0.15 0.11 0.081 0.044 0.035 0.030 0.037 0.0330.027 0.061 0.043 0.15 0.065 0.15 0.088 0.15 0.095 0.15 0.094 0.15 0.10 0.15 0.10 0.072 0.039 0.0410.038 0.032 0.033 0.031 0.031 0 63291 59S.XLS STUDY# 6329-159 F54108-24 F54102-24 FS4097-24 F54071-24 FW93-24 F5409r),-24 F54101-24 F54110-24 F54096-24 FW90-24 FW78-24 FWSS-24 F54106-24 FW89-24 F54087-24 F54080-24 FW92-24 F54109-24 F54114-24 serum bik 1 serum bik2 serum spk 1 serum spk 2 F54113-24 FW82-24 F54111-24 F54098-24 F54084-24 FW79-24 F54116-24 F54113-48 FW82-48 F54111-48 F54098-48 FW84-48 FW79-48 F5411 6-48 F54113-192 F54082-192 FS4111-192 FW98-192 FW79-192 FW84-192 F54116-192 F54113-360 FW82-360 F54111-360 F54098-360 F54079-360 ................... n 0.014 0.013 0.020 0.015 0.014 0.013 0.012 0.011 0.013 0.025 0.013 0.014 0.013 0.014 0.014 0.016 0.016 0.015 0.016 0.039 0.028 0.004 63 0.071 0.004 63 0.082 0.064 0.055 0.063 0.064 0.061 0.059 0.064 0.068 0.066 0.073 0.067 0.091 0.059 0.085 0.080 0.073 0.063 0.067 0.068 0.077 0.065 0.065 0.043 0.063 0.054 0.063 . ............ ......... Va.: L 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 A . ...M. S. 3:@: ............. ....................... UC j-j--a-- ow Ole I'ii 0.28 0.028 0.25 0.025 0.40 0.040 0.30 0.030 0.27 0.027 0.26 0.026 0.24 0.024 0.23 0.023 0.26 0.026 0.50 0.050 0.27 0.027 0.27 0.027 0.26 0.026 0.28 0.028 0.27 0.027 0.32 0.032 0.32 0.032 0.31 0.031 0.32 0.032 0.77 0.077 0.55 0.055 94% 1.4 0.15 0.14 109% 1.6 0.15 0.16 1.3 0.13 1.1 O..ll 1.3 0.13 1.3 0.13 1.2 0.12 1.2 0.12 1.3 0.13 1.4 0.14 1.3 0.13 1.5 0.15 1.3 0.13 1.8 0.18 1.2 0.12 1.7 0.17 1.6 0.16 1.5 0.15 1.3 0.13 1.3 0.13 1.4 0.14 1.5 0.15 1.3 0.13 1.3 0.13 0.87 0.087 1.3 0.13 1.1 0.11 1.3 0.13 0 1 0A- 6329159S.XLS STUDY 6329-159 4 F54084-360 F54116-360 Serum Sik 1 Serum Blk 2 Serum Spk 1 Serum Spk 2 F54082-D22 F54111-D22 F54079-D22 F541 16-D22 FW82-D28 FS4111-D28 F54079-D28 F54116-D28 FW99-4 F54099-8 FW99-12 F54099-24 F54099-48 F54099-192 F54099-3W F541124 F54112-8 F54112-12 F54112-24 F54112-48 F54112-192 F54112-360 SERUM SPIKE-63-1 SERUM SPIKE-63-2 SERUM SPIKE-63-3 SERUM SPIKE-126-1 SERUM SPIKE-126-2 SERUM SPIKE-12r>-3 serum blank-I serum blank-2 serum blank-3 spike63-1 spike63-2 spike63-3 spike634 spike63-5 spike634 spike63-7 spike63-8 Serum bink1 Serum bink2 Serum spike63-1 Senim spike63-2 ..........."a'l"u.a*r@i@@@-": ........ .............................................. U' vrd .. ....... 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 126 0.004 126 0.004 126 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.069 0.069 0.025 0.032 0.069 0.076 0.046 0.041 0.040 0.045 0.048 0.036 0.033 0.036 0.028 0.026 0.074 0.087 0.047 0.033 0.029 0.038 0.059 0.066 0.051 0.066 0.059 0.041 0.059 '0.079 0.074 0.083 0.12 0.12 0.10 0.040 0.019 0.058 0.058 0.092 0.053 0.055 0.058 0.072 0.060 0.045 0.017 0.12 0.21 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 91% 101% 78% 105% 97% 55% 77% 79% 77% 77% 121% 70% 73% 77% 95% 79% 153% 273% 1.4 1.4 0.50 0.65 1.4 l@.5 0.91 0.82 0.81 0.89 0.96 0.71 0.66 0.73 0.55 0.51 1.5 1.7 0.95 0.67 0.57 0.76 1.2 1.3 1.0 1.3 1.2 0.82 1.2 1.6 1.5 1.7 2.3 2.4 2.0 0.79 0.39 1.2 1.2 1.8 1.1 1.1 1.2 1.4 1.2 0.90 0.33 2.3 4.1 0.14 0.14 0.050 0.065 0.15 0.14 0.15 0.15 0.091. 0.082 0.081 0.089 0.10 0.071 0.066 0.073 0.055 0.051 0.15 0.17 0.095 0.067 0.057 0.076 0.12 0.13 0.10 0.13 0.12 0.082 0.15 0.12 0.15 0.16 0.15 0.15 0.30 0.17 0.30 0.23 0.30 0.24 0.20 0.079 0.039 0.15 0.12 0.15 0.15 0.12 0.18 0.15 0.11 0.15 0.15 0.11 0.12 0.15 0.14 0.15 0.12 0.090 0.033 0.15 0.23 0.15 0.41 6329159S.XLS STUDY# 6329-159 . ...... .30 .................................... -nnm Serum blank1 Seruffbnlank2 Serum spike1 Serum spike2 Serum spike3 Swum spow 4 Serum spike5 Serum spike6 Serum spike7 Serum blank3 Serum blank4 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 F54088-48 F5409948 F5408948 F54087-Q F54080-48 F54M248 F54109-48 F5411448 F54088 dy8 F54W9 dyS serum spike8 serum spike9 serum spike10 SERUM BLK 1 SERUM BLK 2 SERUM SPK 1 SERUM SPK 2 SERUM SPK 3 F5Q89-DY8 F54087-DYS 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 F54080-DY8 F54092-DYa F54109-DY8 F54114-DY8 F54OW15 F54099-1 5 F54089-1 5 F54087-1 5 F54NO-15 F54=-15 F54109-15 F54114-15 F54089-D22 F54087-D22 F54109-D22 F54114-D22 F54089-d28 F54087-d28 C..9 Dom- 0.031 0.021 0.074 0.066 0.065 0.060 0.070 0.069 0.068 0.023 0.020 0.019 0.021 0.017 0.020 0.017 0.016 0.022 0.022 0.018 0.014 0.059 0.060 0.060 0.031 0.016 0.056 0.069 0.072 0.026 0.029 0.029 0.036 0.028 0.035 0.010 0.026 0.022 0.021 0.019 0.022 0.025 0.020 0.019 0.018 0.019 0.019 0.018 0.018 ua M orams) 0-.-10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 ..... ............................... . ......... .......... o'1"'EM ug/ ................e.........:..:..,..:.rcvr .F % 2.-6------------------ 0.62 2.0 0.42 0.062 0.042 2.0 98% 1.5 0.15 0.15 2.0 87% 1.3 0.15 0.13 2.0 86% 1.3 0.15 0.13 2.0 80% 2.0 93% 1.2 0.15 0.12 1.4 0.15 0.14. 2.0 91% 1.4 0.15 0.14 2.0 90% 1.4 0.15 0.14 2.0 0.46 0.046 2.0 0.41 0.041 2.0 0.37 0.037 2.0 0.43 0.043 2.0 0.34 0.034 2.0 0.40 0.040 2.0 0.34 0.034 2.0 0.32 0.032 2.0 0.45 0.045 2.0 0.44 0.044 2.0 0.36 0.036 2.0 0.28 0.028 2.0 78% 2.0 79% 1.2 0.15 0.12 1.2 0.15 0.12 2.0 79% 1.2 0.15 0.12 2.0 0.62 0.062 2.0 0.32 0.032 2.0 74% 2.0 91% 2.0 95% 2.0 1.1 1.4 1.4 0.52 0.15 0.15 0.15 0.11 0.14 0.14 0.052 2.0 0.59 0.059 2.0 0.59 0.059 2.0 0.73 0.073 1.o 0.56 0.056 2.0 0.69 0.069 2.0 0.21 0.021 2.0 0.52 0.052 2.0 0.43 0.043 2.0 0.43 0.043 2.0 0.39 0.039 2.0 0.44 0.044 2.0 0.50 0.050 2.0 0.40 0.040 2.0 0.39 0.039 2.0 0.36 0.036 2.0 0.37 0.037 2.0 0.39 0.039 2.0 0.36 0.036 2.0 0.35 0.035 63291 SOS.XLS STUDY 6329-159 F54109-M F54114-d2S SERUM SPIKE 634 SERUM SPIKE 63@-5 SERUM SPIKE 63-6 SERUM SPIKE 63-7 SERUM BLK 1 SERUM BLK 2 SERUM SPK 63-1 SERUM SPK 63-2 SERUM SPK 63-3 F5410548 F5407548 Ai . .. . ...... t0 il*ei@ 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.6 o 0.017 0.031 0.049 0.061 0.059 0.031 0.017 0.061 0.078 0.062 0.021 0.026 3S o.io 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 .. ............ i-nSA-0. ..... n -V..0. M.. U 2.0 2.0 2.0 41% 2.0 65% 2.0 81% 2.0 78% 2.0 2.0 2.0 81% 2.0 103% 2.0 82% 2.0 2.0 0.41 0.33 0.62 0.98 1.2 1.2 0.62 0.34 1.2 1.6 1.2 0.42 0-.52 ...... ac -F-@-.-4--@t-u-@-"-'-F I' 0.041 0.033 0.15 0.062 0.15 0.10 0.15 0.12 0.15 0.12 0.062 0.034 0.15 0.12 0.15 0.16 0.15 0.12 0.042 0.052 6329159S.XLS STUDY# 6329-159 SERUM ............ ................ F.r,.95". .95 . ... ......................... .............1.:.1...@.--.l@.'..@..-...'.@..@".'.'...k'..."..d'..-..'.@5P.1, serum blank-1 serum blank-2 serum spike 63-1 0.004 63 serum spike63-2 0.004 63 serum spike 94.5-1 0.004 94.5 serum spike 94.5-2 0.004 serum spike 126-1 0.004 94.5 126 serum spike 126-2 0.004 126 serum spike 126-3 0.004 126 serum spike 126-4 0.004 126 F54081-4 F54075-4 F54107-4 F54105-4 F54074-4 Blank 1 Blank 2 Blank 3 Blank 4 Blank 5 Blank 6 Serum Spk-1 0.004 63 Serum Spk-2 0.004 63 Serum Spk-3 0.004 63 Serum Spk-4 0.004 126 Serum Spk-5 0.004 126 Serum Spk-6 0.004 126 Serum Spk-7 0.004 126 Serum Spk-8 0.004 126 Serum Spk-9 0.004 126 Serum Spk-10 0.004 126 F54085-4 F54086-4 F54104-4 F54077-4 F54083-4 F54094-4 F54100-4 F54091-4 serum bik 1 serum bik 2 serum spk 1 0.004 63 serum spk 2 0.004 63 F54097-4 F54102-4 F54108-4 F54071-4 . ....... d' ........ ..... F 0.036 0.020 0.070 0.070 0.063 0.046 0.15 0.099 0.15 0.13 0.040 0.032 0.024 0.025 0.034 0.71 0.34 0.36 0.084 0.038 0.039 0.055 0.068 0.073 0.10 0.11 0.11 0.11 0.098 0.12 0.10 0.045 0.027 0.025 0.021 0.020 0.021 0.020 0.019 0.029 0.024 0.071 0.072 0.022 0.020 0.016 0.017 ........... TiSA- g. .n.... .. . ... ...... . ............... 0.10 2.0 0.10 2.0 0.10 2.0 93% 0.10 0.10 0.10 2.0 92% 2.0 56% 2.0 41% 0.10 2.0 101% 0.10 2.0 65% 0.10 2.0 98% 0.10 2.0 85% 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 0.10 2.0 73% 2.0 90% 0.10 0.10 2.0 96% 2.0 67% 0.10 2.0 74% 0.10 0.10 0.10 0.10 2.0 73% 2.0 72% 2.0 65% 2.0 77% 0.10 2.0 68% 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 0.10 2.0 94% 2.0 95% 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 @iA ..... P ND ND 1.4 1.4 1.3 0.92 3.1 2.0 3.0 2.6 ND ND ND ND ND 14 6.9 7.2 1.7 ND ND 1.1 1.4 1.5 2.0 2.2 2.2 2.2 2.0 2.3 2.1 ND ND ND ND ND ND ND ND ND ND 1.4 1.4 ND ND ND ND F 0.15 0.15 0.23 0.23 0.30 0.30 0.30 0.30 0.15 0.15 0.15 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.15 0.15 0.14 0.14 0.13 0.092 0.31. 0.20 0.30 0.26 ND ND ND ND ND 1.4 0.69 0.72 0.17 ND ND 0.11 0.14 0.15 0.20 0.22 0.22 0.22 0.20 0.23 0.21 ND ND ND ND ND ND ND ND ND ND 0.14 0.14 ND ND ND ND 6329159S.XLS STUDY# 6329-159 SERUM ................... .......... . .......... ........................A..'.... C-91 ........... ..................... . .............. ................ pp .................. 4m F54093-4 0.020 0.10 2.0 F54095-4 0.016 0.10 2.0 F54101-4 0.015 0.10 2.0 F54078-4 0.021 0.10 2.0 F54090-4 0.020 0.10 2.0 F54096-4 0.017 0.10 2.0 F54110-4 0.020 0.10 2.0 F54087-4 0.025 0.10 2.0 F54088-4 0.028 0.10 2.0 F54089-4 0.020 0.10 2.0 F54106-4 0.032 0.10 2.0 F54080-4 0.025 0.10 2.0 F54092-4 0.019 0.10 2.0 F54109-4 0.021 0.10 2.0 F54114-4 0.039 0.10 2.0 F54076-4 0.041 0.10 2.0 F54082-4 0.057 0.10 2.0 F54111-4 0.096 0.10 2.0 F54113-4 0.082 0.10 2.0 F54079-4 0.12 0.10 2.0 F54084-4 0.067 0.10 2.0 F54098-4 0.046 0.10 2.0 F54116-4 0.10 0.10 2.0 F54075-8 0.025 0.10 2.0 F54081-8 0.020 0.10 2.0 F54105-8 0.021 0.10 2.0 F54107-8 0.015 0.10 2.0 F54074-8 0.015 0.10 2.0 F54085-8 0.021 0.10 2.0 F54086-8 0.031 0.10 2.0 serum bik 1 0.029 0.10 2.0 serum blk2 0.028 0.10 2.0 serum spk 1 0.004 63 0.080 0.10 2.0 106% serum spk 2 0.004 63 0.069 0.10 2.0 91% F54104-8 0.031 0.10 2.0 F54077-8 0.023 0.10 2.0 F54083-8 0.018 0.10 2.0 F54094-8 0.025 0.10 2.0 F541 00-8 0.017 0.10 2.0 F54091-8 0.014 0.10 2.0 F54097-8 0.014 0.10 2.0 F54102-8 0.013 0.10 2.0 F54108-8 0.013 0.10 2.0 F54071- 8 0.015 0.10 2.0 F54093 - 8 0.019 0.10 2.0 F54095 - 8 0.026 0.10 2.0 F54101- 8 0.016 0.10 2.0 F54078-8 0.021 0.10 2.0 p .. ....... @ka ND ND ND ND ND ND ND ND ND ND ND 'ND ND ND ND ND 1.1 1.9 1.6 2.3 1.3 ND 2.0 ND ND ND ND ND ND ND ND ND 1.6 0.15 1.4 0.15 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.11 0.19 0.16 0.23 0.13 ND 0.20 ND ND ND ND ND ND ND ND ND 0.16 0.14 ND ND ND ND ND ND ND ND ND ND ND ND ND ND 6329159S.XLS STUDY# 6329-159 SERUM 30 F54090- 8 F54096-8 F54110- 8 F54087-8 F54088- 8 F54089-8 F54080-8 F54106- 8 F54092-8 F54109- 8 F54114- 8 F54076-8 F54082- 8 F54111- 8 F54113- 8 F54079-8 F54084-8 F54098- 8 F54116- 8 serum bik 1 serum bik2 serum spk 1 serum spk 2 F54075-12 F54081-12 F54105-12 F54107-12 F54074-12 F54085-12 F54086-12 F54086-12 F54077-12 F54083-12 F54094-12 F54100-12 F54091-12 F54097-12 F54102-12 F54108-12 F54077-12 F54071-12 F54093-12 F54095-12 F54101-12 F54110-12 F54096-12 F54090-12 F54078-12 'O'h FC. ..............sd 0.004 63 0.004 63 y t ............ --IMSA 0.021 0.10 2.0 0.017 0.10 2.0 0.075 0.10 2.0 0.022 0.10 2.0 0.033 0.10 2.0 0.031 0.10 2.0 0.032 0.10 2.0 0.043 0.10 2.0 0.037 0.10 2.0 0.024 0.10 2.0 0.034 0.10 2.0 0.045 0.10 2.0 0.063 0.10 2.0 0.044 0.10 2.0 0.048 0.10 2.0 0.042 0.10 2.0 0.045 0.10 2.0 0.050 0.10 2.0 0.17 0.10 2.0 0.012 0.10 2.0 0.018 0.10 2.0 0.066 0.10 2.0 0.070 0.10 2.0 0.028 0.10 2.0 0.021 0.10 2.0 0.026 0.10 2.0 0.019 0.10 2.0 0.017 0.10 2.0 0.018 0.10 2.0 0.020 0.10 2.0 0.025 0.10 2.0 0.038 0.10 2.0 0.032 0.10 2.0 0.035 0.10 2.0 0.022 0.10 2.0 0.029 0.10 2.0 0.025 0.10 2.0 0.022 0.10 2.0 0.014 0.10 2.0 0.046 0.10 2.0 0.021 0.10 2.0 0.016 0.10 2.0 0.016 0.10 2.0 0.020 0.10 2.0 0.016 0.10 2.0 0.012 0.10 2.0 0.017 0.10 2.0 0.014 0.10 2.0 g! 87% 93% .. ... --M ..........P.!.k..-"..'... to- ND ND ND ND 1.5 0.15 ND ND. ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1.3 0.13 ND ND ND ND ND ND ND ND 1.0 0.10 3.4 0.34 ND ND ND ND 1.3 0.15 0.13 1.4 0.15 0.14 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND N@D ND ND ND ND 6329159SXLS STUDY# 6329-159 SERUM ................. ........ ituti.o.. . D..'.......................................p..k..d. ............... a -F@ ............... k" F54088-12 F54106-12 F54089-12 F54087-12 F54080-12 F54092-12 F54109-12 F54114-12 F54076-12 F54113-12 0.025 0.10 2.0 ND ND 0.027 0.10 2.0 NO ND 0.019 0.10 2.0 ND ND 0.019 0.10 2.0 ND ND 0.018 0.10 2.0 ND ND 0.017 0.10 2.0 ND ND 0.021 0.10 2.0 ND ND 0.026 0.10 2.0 ND ND 0.027 0.10 2.0 ND ND 0.022 0.10 2.0 ND ND F54082-12 F54111-12 F54098-12 F54084-12 F54079-12 0.034 0.10 2.0 ND ND 0.046 0.10 2.0 ND ND 0.042 0.10 2.0 ND ND 0.044 0.10 2.0 ND ND 0.043 0.10 2.0 ND ND F54116-12 Serum Blk-1 Serum Bik-2 Serum Spk-1 Serum Spk-2 Serum Spk-3 Serum Spk-4 Serum Spk-5 Serum Spk-6 Serum Spk-7 Serum Spk-8 F54105-24 F54075-24 F54081-24 F54107-24 F54104-24 F54086-24 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.004 63 0.047 0.033 0.030 0.034 0.040 0.050 0.054 0.083 0.053 0.049 0.055 0.041 0.022 0.017 0.015 0.018 0.016 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 2.0 2.0 2.0 2.0 44% 2.0 52% 2.0 66% 2.0 72% 2.0 110% 2.0 71% 2.0 65% 2.0 73% 2.0 2.0 2.0 2.0 2.0 2.0 ND ND ND 0.67 0.79 0.99 1.1 1.7 1.1 0.99 1.1 ND ND ND ND ND ND 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 ND ND ND 0.067 0.079 0.10 0.11 0.17 0.11 0.10 0.11 ND ND ND ND ND ND F54074-24 Blank 1 Blank 2 spike 1 spike2 spike3 0.004 63 0.004 63 0.004 63 0.013 0.031 0.022 0.033 0.044 0.047 0.10 0.10 0.10 0.10 0.10 0.10 2.0 2.0 2.0 2.0 43% 2.0 58% 2.0 63% ND ND ND 0.65 0.88 0.95 0.15 0.15 0.15 ND ND ND 0.065 0.088 0.095 spike4 spike5 .0.004 63 0.004 63 0.047 0.052 0.10 0.10 2.0 62% 2.0 68% 0.94 1.0 0.15 0.094 0.15 0.10 spike6 0.004 63 0.051 0.10 2.0 67% 1.0 0.15 0.10 Blank 3 Blank4 0.036 0.10 2.0 ND ND 0.020 0.10 2.0 ND ND F54085-24 F54083-24 F54100-24 F54077-24 F54094-24 F54091-24 0.021 0.10 2.0 ND ND 0.019 0.10 2.0 ND ND 0.016 0.10 2.0 ND ND 0.017 0.10 2.0 ND ND 0.015 0.10 2.0 NO ND 0.016 0.10 2.0 ND ND 6329159S.XLS . ....... .. ...........................4........Y........ ....... .F..CG...S...............o.u.r..:...:.. ip, ............ ...................................Ip.i.n.::r:ea:oi .. ............... F54108-24 F54102-24 F54097-24 F54071-24 F54093-24 F54095-24 F54101-24 F54110-24 F54096-24 F54090-24 F54078-24 F54088-24 F54106-24 F54089-24 F54087-24 F54080-24 F54092-24 F54109-24 F54114-24 serum bik 1 serum bik2 serum spk 1 serum spk 2 F54113-24 F54082-24 F54111-24 F54098-24 F54084-24 F54079-24 F54116-24 F54113-48 F54082-48 F54111-48 F54098-48 F54084-48 F54079-48 F54116-48 F54113-192 F54082-192 F54111-192 F54098-192 F54079-192 F54084-192 F54116-192 F54113-360 F54082-360 F54111-360 F54098-360 F54079-360 0.014 0.,10 2.0 0.013 0.10 2.0 0.020 0.10 2.0 0.015 0.10 2.0 0.014 0.10 2.0 0.013 0.10 2.0 0.012 0.10 2.0 0.011 0.10 2.0 0.013 0.10 2.0 0.025 0.10 2.0 0.013 0.10 2.0 0.014 0.10 2.0 0.013 0.10 2.0 0.014 0.10 2.0 0.014 0.10 2.0 0.016 0.10 2.0 0.016, 0.10 2.0 0.015 0.10 2.0 0.016 0.10 2.0 0.039 0.10 2.0 0.028 0.10 2.0 0.004 63 0.004 63 0.071 0.10 0.082 0.10 0.064 0.10 2.0 94% 2.0 109% 2.0 0.055 0.10 2.0 0.063 0.10 2.0 0.064 0.10 2.0 0.061 0.10 2.0 0.059 0.10 2.0 0.064 0.10 2.0 0.068 0.10 2.0 0.066 0.10 2.0 0.073 0.10 2.0 0.067 0.10 2.0 0.091 0.10 2.0 0.059 0.10 2.0 0.085 0.10 2.0 0.080 0.10 2.0 0.073 0.10 2.0 0.063 0.10 2.0 0.067 0.10 2.0 0.068 0.10 2.0 0.077 0.10 2.0 0.065 0.10 2.0 0.065 0.10 2.0 0.043 0.10 2.0 0.063 0.10 2.0 0.054 0.10 2.0 0.063 0.10 2.0 STUDY# 6329-159 SERUM . .............. ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND. 1.4 0.15 0.14 1.6 0.15 0.16 1.3 0.13 1.1 0.11 1.3 0.13 1.3 0.13 1.2 0.12 1.2 0.12 1.3 0.13 1.4 0.14 1.3 0.13 1.5 0.15 1.3 0.13 1.8 0.18 1.2 0.12 1.7 0.17 1.6 0.16 1.5 0.15 1.3 0.13 1.3 0.13 1.4 0.14 1.5 0.15 1.3 0.13 1.3 0.13 ND 0.087 1.3 0.13 1.1 0.11 1.3 0.13 JL 6329159S.XLS STUDY# 6329-159 SERUM ...................................................... .. ............. ............. .. F54084-360 F54116-360 Serum Bik 1 Serum Blk2 Serum Spk 1 Serum Spk 2 F54082-D22 F541 11-D22 F54079-D22 F54116-D22 F54082-D28 F54111-D28 F54079-D28 F54116-D28 F54099-4 F54099-8 F54099-12 F54099-24 F54099-48 F54099-192 F54099-360 F54112-4 F54112-8 F54112-12 F@54112-24 F54112-48 F54112-192 F54112-360 SERUM SPIKE-63-1 SERUM SPIKE-63-2 SERUM SPIKE-63-3 SERUM SPIKE-126-1 SERUM SPIKE-126-2 SERUM SPIKE-126-3 serum blank-I serum blank-2 serum blank-3 spike63-1 spike63-2 spike63-3 spike63-4 spike63-5 spike63-6 spike63-7 spike63-8 Serum blnk 1 Serum blnk2 Serum spike63-1 Serum spike63-2 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 ... .....'.d. ... .......P.PM 0 0.069 0.025 0.032 63 0.069 63 0.076 0.046 0.041 0.040 0.045 0.048 0.036 0.033 0.036 0.028 0.026 0.074 0.087 0.047 0.033 0.029 0.038 0.059 0.066 0.051 0.066 0.059 0.041 63 0.059 63 0.079 63 0.074 126 0.083 126 0.12 126 0.12 0.10 ().040 0.019 63 0.058 63 0.058 63 0.092 63 0.053 63 0.055 63 0.058 63 0.072 83 0.060 0.045 0.017 63 0.12 63 0.21 ........... I....-.ISA ---------0 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 A**' I........i ............. u 1.4 1.4 ND ND 91% 1.4 0.15 101% 1.5 0.15 ND ND ND ND ND ND ND ND ND ND 1.5 1.7 ND ND ND ND 1.2 1.3 1.0 1.3 1.2 ND 78% 1.2 0.15 105% 1.6 0.15 97% 1.5 0.15 55% 1.7 0.30 77% 2.3 0.30 79% 2.4 0.30 2.0 ND ND 77% 1.2 0.15 77% 1.2 0.15 121% 1.8 0.15 70% 1.1 0.15 73% 1.1 0.15 77% 1.2 0.15 95% 1.4 0.15 79% 1.2 0.15 ND ND 153% 2.3 0.15 273% 4.1 0.15 0.14 0.14 ND ND 0.14 0.15 ND ND ND ND ND ND ND ND ND ND 0.15 0.17 ND ND ND ND 0.12 0.13 0.10 0.13 0.12 ND 0.12 0.16 0.15. 0.17 0.23 0.24 0.20 ND ND 0.12 0.12 0.18 0.11 0.11 0.12 0.14 0.12 ND ND 0.23 0.41 6329159S.XLS STUDY# 6329-159 SERUM ........... ...................... ............ Serubmlan1k Serum blank2 Serum spike1 Serum spike2 Serum spike3 Serum spike4 Serum spike5 Serum spike6 Serum spike7 Serum blank3 Serum blank4 F54088-48 F54099-48 F54089-48 F54087-48 F54080-48 F54092-48 F54109-48 F54114-48 F54088 dy8 F54099 dy8 serum spike8 serum spike9 serum spike10 SERUM BLK 1 SERUM BLK 2 SERUM SPK 1 SERUM SPK 2 SERUM SPK 3 F54089-DY8 F54087-DY8 F54080-DYB F54092-DYS F54109-DY8 F54114-DY8 F54088-15 F54099-15 F54089-15 F54087-15 F54080-15 F54092-15 F54109-15 F54114-1 5 F54089-D22 F54087-D22 F54109-D22 F54114-D22 F54089-d28 F54087-d28 .........o..n...................... :9@ .... ... Iu"ti"o"n".:.@.:..:.:.i.r.de.ie. -it pi -P ...... --6.-031 0.021 0.004 63 0.074 0.004 63 0.066 0.004 83 0.065 0.004 63 0.060 0.004 83 0.070 0.004 63 0.069 0.004 63 0.068 0.023 0.020 0.019 0.021 0.017 0.020 0.017 0.016 0.022 0.022 0.018 0.014 0.004 63 0.059 0.004 63 0.060 0.004 63 0.060 0.031 0.016 0.004 63 0.056 0.004 63 0.069 0.004 63 0.072 0.026 0.029 0.029 0.036 0.028 0.035 0.010 0.026 0.022 0.021 0.019 0.022 0.025 0.020 0.019 0.018 0.019 0.019 0.018 0.018 .. ........ ......... -c 0) ua:.Um 0.10 2.0 0.10 2.0 0.10 2.0 98% 0.10 2.0 87% 0.10 2.0 86% 0.10 2.0 80% 0.10 2.0 93% 0.10 2.0 91% o.io 2.0 90% 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 o.io 2.0 0.10 2.0 0.10 2.0 78% 0.10 2.0 79% 0.10 2.0 79% 0.10 2.0 0.10 2.0 0.10 2.0 74% 0.10 2.0 91% 0.10 2.0 95% 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 ipp. lit'sam- ND ND -- 66' 1.5 1.3 1.3 1.2 1.4 1.4 1.4 ND ND ND ND ND ND ND ND ND ND ND ND 1.2 1.2 1.2 ND ND 1.1 1.4 1.4 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 ... ND ND 0.15 0.13 0.13 0.12 0.14 0.14 0.14 ND ND ND ND ND ND ND ND ND ND ND ND 0.12 0.12 0.12 ND ND 0.11 0.14 0.14 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND law 63291 59S.XLS STUDY# 6329-159 SERUM ....................................... ........... ................... F54109A2$ -------F54114-d28 SERUM SPIKE 63-4 0.004 63 SERUM SPIKE 63-5 0.004 63 SERUM SPIKE 63-6 0.004 63 SERUM SPIKE 63-7 0.004 63 SERUM BLK 1 SERUM BLK 2 SERUM SPK 63-1 0.004 63 SERUM SPK 63-2 0.004 63 SERUM SPK 63-3 0.004 63 F5410548 F54075-48 6.620 0.017 0.031 0.049 0.061 0.059 0.031 0.017 0.081 0.078 0.062 0.021 0.026 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 2.0 2.0 2.0 41% 2.0 65% 2.0 81% 2.0 78% 2.0 2.0 2.0 81% 2.0 103% 2.0 82% 2.0 2.0 ND ND 0.62 0.98 1.2 1.2 ND ND 1.2 1.6 1.2 ND ND .... ....... :::::rvcr. 0.15 0.15 0.15 0.15 0.15 0.15 0.15 ND ND 0.062 0.10 0.12 0.12 ND ND 0.12 0.16 0.12 ND ND Control F54074 F54075 F54081 F54085 F54086 F54104 F54105 F54107 5 mgfkg F54077 F54083 F54091 F54094 F54097 F54 100 F54102 F54108 10 mg/kg F54071 F54093 F54095 F54101 F54078 F54090 F54096 F541 10 100 mglkg F54080 F54087 F54088 F54089 F54092 F54099 F54106 F54109 F54114 500 mgtkg F54076 F54079 F54082 F54084 F54098 F541 11 F54112 F54113 F54116 HWI 6329-159,AMDT 042095.1 ppm F- inserum 4 hour 0.670 0.645 0.800 0.898 0.542 0.492 0.509 0.484 8 hour 0.304 0.490 0.398 0.428 0.616 0.616 0.426 0.304 12 hour 0.346 0.558 0.416 0.366 0.408 0.502 0.528 0.388 24 hour 0.268 0.440 0.346 0.412 0.328 0.366 0.812 0.304 Summary ofOrion serum analysiswithout entering"ND". This summary isused to determinetherelativhealflifet,hereforeit Isappropriateto use valueswhich are less than the detectionBmit 4 hour 0.416 0.400 0.384 0.418 0.446 0.408 0.406 0.318 8 hour 0.458 0.352 0.282 0.506 0.278 0.346 0.258 0.254 12 hour 0.839 0.636 0.576 0.708 0.492 0.442 0.436 0.288 24 hour 0.330 0.380 0.314 0.308 0.400 0.324 0.250 0.278 4 hour 0.336 0.408 0.316 0.290 0.412 0.390 0.336 0.392 8 hour 0.302 0.374 0.514 0.322 0.424 0.424 0.342 1.500 12 hour 0.412 0.328 0.320 OA04 0.284 0.342 0.246 0.312 24 hour 0.302 0.274 0.256 0.240 0.266 0.500 0.264 0.226 4 hour 0.508 0.496 0.552 0.406 0.376 0.552 0.6'JO 0.418 0.776 8 hour 0.638 0.438 0.662 0.624 0.740 0.514 0.860 0.488 0.676 12 hour 0.358 0.372 0.508 0.380 0.332 1.478 0.536 0.410 0.524 24 hour 0.320 0.270 0.274 0.278 0.322 1.748 0.256 0.306 0.322 48 hour 192hour 360 hour 528hour 672 hour 0.398 0.336 0.588 0.524 0.426 0.434 0.362 0.390 0.350 0.360 0.948 0.446 0.438 0.666 0.564 0.694 0.572 0.500 0.400 0.374 0.390 0.406 0.330 4 hour 0.810 2.320 1.130 1.344 0.926 1.914 0.758 1.638 2.040 8 hour 0.890 0.838 1.254 0.890 1.000 0.878 1.184 0.954 3.440 12 hour 0.546 0.852 0.686 0.870 0.836 0.912 1.310 0.436 0.940 24 hour 1.170 1.104 1.222 1.276 1.252 1.022 1.276 1.286 48 hour 192hour 360 hour 528 hour 672 hour 1.184 1.318 1.824 1.332 1.452 1.314 1.364 1.706 1.364 1.459 1.546 1.344 1.256 1.184 1.596 1.292 1.266 0.866 1.370 1.078 1.252 0.822 1.292 1.380 0.808 0.912 0.822 0.890 0.660 0.964 0.710 0.728 0 rz CL. 904.24 09 Figure2 ppm F- in Serum (Group 4) o.i 0 100 200 300 400 500 Time (hoursPostDose) 10.000- Figure 1 ppm F- in serum (Group 5) 0 OD 1.000 tl/2(48hour-872 hour) 28 Days 0.100 0 100 200 300 400 500 Time (HoursPostDose) 9.11.4Summary and raw data;ppm F-inserum as determinedby thermalextractiofnollowedby analysisusing Skalarsegmented flow analyzerwithion selectiveelectrode. ooolL7t:OL,t BE: 6329-159 SERUM SRMPLES RMDT 42095.1 Date of Analysis: May 4, 8,9, 11,24 and May Analyst: DOW 25, 1995 The samples are burned in the Dohrman at 950 C using 0.10 mL of the serum. The gas iscollectedin2.0 mL of 1:1TISFIR/Milli-O water. The samples are then analyzed on a Skalar Segmented Flow Rnalyzer using the Ion SpecificElectrode (ISE)Method. TISRB buffer isadded to each sample as itproceeds through the system. The sample then goes through a heated mixing coil before the potentialbetween the ion selectiueelectrodeand the reference electrode ismeasured. The signalisamplifiedand relatedto the fluorideconcentration. The instrument was calibratedin the ranges of 0.015 - 0.15 PPM and 0.15 - 1.50 ppm fluoride.The standard curue for the high range was plotted using the inuerse logarithm option.The standard curue for the low range islinear.Rilstandards and samples were then calculatedby the Skalar software using these curues. Rilresultsbelow 0.0001 ppm appear on the raw data as #.####. R qualitycontrolstandard was analyzed euery 10 samples to check for accuracy and drift. Raw data istaken from the appropriatecalibratedrange of the Skalar printoutand summarized on an Excel spreadsheet. The finalresultsare adjusted for the collectionuolume and any sub uent dilutions. -A SUMMARY of6329-159 SERUM SAMPLES AMI)T 42095.1 GROUP 1 Dose Level: 0 F54074 F54075 F54080 F54085 F54085 F54086 F54104 F54105 F54107 0.51 0.61 0.84 0.98 0.58 0.34 0.46 0.35 0.64 0.40 0.62 0.73 0.55 0.31 0.42 0.63 0.43. 0.29 0.70 0.81 0.60 0.40 0.70 0.47 0.40 0.00 0.31 0.35 0.47 1.08 0.31 0.52 0.36 F54077 0.53 0.58 1.22 0.34 F54077 0.39 F54083 0.33 0.43 0.98 0.00 F54083 0.42 GROUP 2 F54091 0.36 0.12 0.90 0.28 Dose Level :5 mg/kg F54094 0.36 0.45 1.03 0.27 F54097 0.40 0.05 0.67 0.48 F541 00 0.39 0.41 0.62 0.00 F541 00 0.31 F54102 0.38 0.29 0.46 0.18 F54108 0.20 0.01 0.20 0.27 F54071 0.24 0.16 0.00 0.24 F54078 0.39 0.22 0.32 0.52 F54090 0.28 0.20 0.49 1.02 GROUP3 F54093 0.43 0.29 0.00 0.21 Dose Level: 10 mgtkg F54095 0.24 0.41 0.00 0.42 F54096 0.13 0.00 0.04 0.23 F541 01 0.15 0.13 0.00 0.34 F541 10 0.25 1.85 0.00 0.30 F54080 0.56 0.73 0.53 1.21 0.28 F54080 0.57 F54087 0.50 0.41 0.64 0.81 0.39 F54088 0.40 0.64 0.79 0.48 0.31 GROUP4 F54089 0.36 0.75 0.65 0.99 0.22 Dose Level: 100 mg/kg F54092 0.28 0.87 0.44 1.02 0.19 F54099 0.63 0.60 1.79 2.15 1.14 F54099 0.47 F54106 0.68 1.05 0.96 0.88 F54109 0.32 0.67 0.64 0.78 0.43 F54114 0.96 0.90 0.72 0.64 0.46 0.65 0.66 0.32 0.49 0.73 0.74 0.34 0.56 0.73 0.21 0.35 0.00 0.33 0.43 0.61 0.53 0.50 0.29 0.27 0.32 0.29 0.34 0.34 0.23 0.34 0.36 Page 1 elf SLTMMARY SERUM AAMT of 6329-159 SAMIPLES 42095.1 F54076 0.92 1.17 0.68 F54079 2.94 1.36 1.25 1.00 1.05 1.28 1.36 1.07 0.86 F54082 1.40 1.75 0.95 0.86 1.37 1.28 0.71 1.17 1.21 GROUPS F54084 1.78 1.44 1.30 1.03 1.73 1.46 1.40 Dose Level: 500 mgtkg F54098 1.25 1.58 1.39 1.10 1.37 1.25 1.04 F54111 2.42 1.19 1.40 1.18 1.52 1.02 1.22 1.14 0.89 F54112 1.05 1.47 1.65 1.25 1.63 1.45 0.90 F54113 2.23 1.40 0.66 1.17 1.44 1.55 1.17 F5411-6 2.61 4.56 1.32 1.20 1.69 1.15 1.92 1.09 1.00 Page 2 of.)014ri HWI-1 59A.XLS 1995-06-2110:11 OutPut of:950504Al Operator : DDW Date ofthe Analysis:1995-05-0408:18 AnalysisFileName :C:\SKA-LAR\DATA\IIWIDATA\SERUM\950504Al I Tracer 1.50 1.46 97% 2 Drift 1.50 1.47 98% 3 Wash 0.00 4 Standard 1 0.015 0.015 100% 5 Standard2 0.03 0.03 101% 6 Standard3 0.06 0.06 99% 7 Standard4 0.09 0.09 99% 8 Standard5 0.12 0.12 102% 9 Standard6 0.15 0.15 99% 10 Standard7 0.30 0.29 96% 11 Standard8 0.60 0.61 102% 12 Standard9 1.20 1.23 102% 13 Standard 10 1.50 1.47 98% 14 Drift 1.50 1.47 98% 15 Wash 0.00 16 Blk 1 0.04 17 Blk 2 0.02 18 Spk 63-1 0.07 19 Spk 63-2 0.07 20 Spk 94.5-1 0.07 21 Spk 94.5-2 0.04 22 Spk 126-1 0.17 23 Spk 126-2 0.09 24 Spk 126-3 0.13 25 Spk 126-4 0.14 26 Drift 1.50 1.43 95% 27 Wash 0.00 28 F54081-4 0.04 29 F54075-4 0.03 30 F54107-4 0.02 31 F54105-4 0.02 2.0 0.10 0.77 2.0 0.10 0.48 2.0 0.10 1.37 0.004 63.00 0.1 2.0 0,10 1.47 0.004 63.00 0.1 2.0 0.10 1.46 0.004 94.50 0.2 2.0 0.10 0.87 0.004 94.50 0.2 2.0 0.10 3.42 0.004 126.00 0.3 2.0 0.10 1.87 0.004 126.00 0.3 2.0 0.10 2.69 0.004 126.00 0.3 2.0 0.10 2.86 0.004 126.00 0.3 2.0 0.10 0.84 2.0 0.10 0.61 2.0 0.10 0.46 2.0 0.10 0.34 Page 1 HWI-1 59A.XLS 32 F54074-4 0.03 33 Blk-1 0.83 34 Blk-2 0.40 35 Spk 63-1 0.08 36 Spk 63-2 0.09 37 Spk 63-3 0.10 38 Drift 1.50 1.46 98% 39 Wash 0.00 40 Spk 126-1 0.14 41 Spk 126-2 0.14 42 Spk 126-3 0.14 43 Spk 126-4 0.15 44 Spk 126-5 0.13 45 Spk 126-6 0.15 46 Spk 126-7 0.12 47 F54085-4 0.05 48 F54080-4 0.03 49 F54104-4 0.03 50 Drift 1.50 1.47 98% 51 Wash 0.00 52 F54077-4 0.03 53 F54083-4 0.02 54 F54094-4 0.02 55 F54100-4 0.02 56 F54091-4 0.02 57 Blk 1 0.02 58 Blk 2 0.01 59 Spk 1 0.08 60 Spk 2 0.08 61 F54097-4 0.02 62 Drift 1.50 1.44 96% 63 Wash - 0.00 64 F54102-4 0.02 65 F54092-4 0.01 66 F54080-4 0.03 67 F54106-4 0.03 2.0 0.10 0.51 2.0 0.10 16.56 2.0 0.10 8.04 2.0 0.10 1.53 0.004 63.00 0.15 2.0 0.10 1.86 0.004 63.00 0.15 2.0 0.10 1.93 0.004 63.00 0.15 2.0 0.10 2.79 0.004 126.00 0.30 2.0 0.10 2.83 0.004 126.00 0.30 2.0 0.10 2.74 0.004 126.00 0.30 2.0 0.10 2.93 0.004 126.00 0.30 2.0 0.10 2.58 0.004 126.00 0.30 2.0 0.10 3.07 0.004 126.00 0.30 2.0 0.10 2.36 2.0 0.10 0.98 2.0 0.10 0.56 2.0 0.10 0.58 2.0 0.10 0.53 2.0 0.10 0.33 2.0 0.10 0.36 2.0 0.10 0.39 2.0 0.10 0.36 2.0 0.10 0.44 2.0 0.10 0.26 2.0 0.10 1.60 2.0 0.10 1.67 2.0 0.10 0.40 2,0 0.10 0.38 2.0 0.10 0.28 2.0 0.10 0.57 2.0 0,10 0.68 Page 2 HWI-1 59A.XLS 68 F5U689-4 0.02 69 F54"88-4 0.02 70 F54lt87-4 0.02 71 F54YI 104 0.01 72 F54096-4 0.01 73 F540904 0.01 74 Drift 1.50 1.46 97% 75 Wash 0.00 76 F540784 0.02 77 F541014 0.01 78 F540954 0.01 79 F540934 0.02 80 F540714 0.01 81 F54108-4 0.01 82 F54116-4 0.13 83 F540984 0.06 84 F540844 0.09 85 F54079-4 0.15 86 Drift 1.50 1.48 99% 87 Wash 0.00 88 F54113-4 0.11 89 F541114 0.12 90 F540824 0.07 91 F54076-4 0.05 92 F541144 0.04 93 F541094 0.02 94 Drift 1.50 1.47 98% 95 Wash 0.00 2.0 0.10 0.36 2.0 0.10 0.40 2.0 0.10 0.50 2.0 0.10 0.25 2.0 0.10 0.13 2.0 0.10 0.28 2.0 0.10 0.39 2.0 0,10 0.15 2.0 0.10 0.24 2.0 0.10 0.43 2.0 0.10 0.24 2.0 0.10 0.20 2.0 0.10 2.61 2.0 0.10 1.25 2.0 0.10 1.78 2.0 0.10 2.94 2.0 0.10 2.23 2.0 0.10 2.42 2.0 0.10 1.40 2.0 0,10 0.92 2.0 0.10 0.86 2.0 0.10 0.32 T 6 OACK&O Page 3 HWI-1 59B.XLS 1995-06-21 10:15 OutPutof: 950504B I Operator :DDW Date oftheAnalysis: 1995-05-0413:07 AnalysisFileName :C:\SKALAR\DATA\HWIDATA\SERUM\950504BI 1 Tracer 1.50 1.47 98% 2 Ddft 1.50 1.47 98% 3 Wash 0.00 4 Standard 1 0.015 0.014 93% 5 Standard 2 0.03 0.03 104% 6 Standard 3 0.06 0.06 103% 7 Standard 4 0.09 0.09 97% 8 Standard 5 0.12 0.12 101% 9 Standard 6 0.15 0.15 100% 10 Standard 7 0.30 0.29 97% 11 Standard 8 0.60 0.61 101% 12 Standard 9 1.20 1.23 102% 13 Standard 10 1.50 1.48 98% 14 Ddft 1.50 1.45 97% 15 Wash 16 F54075-8 17 F54081-8 18 F54105-8 19 F54107-8 20 F54074-8 21 F54085-8 22 F54086-8 23 BLK-1 24 BLK-2 25 SPK 1 0.00 0.03 0.02 0.03 0.02 0.02 0.03 0.04 0.02 0.07 0.10 2.0 0.10 0.64 2.0 0.10 0.40 2.0 0.10 0.55 2.0 0.10 0.31 2.0 0.10 0.35 2.0 0.10 0.62 2.0 0.10 0.71 2.0 0.10 0.42 2.0 0.10 1.33 2.0 0.10 2.00 0.004 63.00 0.15 26 Drift 1.50 1.44 96% 27 Wash 0.00 28 SPK 2 0.08 29 F54104-8 0.04 30 F54077-8 0.03 31 F54083-8 0.02 2.0 0.10 1.55 2.0 0.10 0.73 2.0 0.10 0.58 2.0 0.10 0.43 Page 1 -3 HWI-1 59B.XLS 32 F54094-8 0.02 2.0 0.10 0.45 33 F54100-8 0.02 2.0 0.10 0.41 34 F54091-8 0.01 2.0 0.10 0.12 35 F54097-8 0.00 2.0 0.10 0.05 36 F54102-8 0.01 2.0 0.10 0.29 37 F54108-8 0.00 2.0 0.10 0.01 38 Drift 1.50 1.40 93% 39 Wash 40 F54071-8 41 F54093-8 42 F54095-8 43 F54101-8 44 F54078-8 45 F54090-8 46 F54096-8 47 F54110-8 48 F54087-8 49 F54088-8 0.00 0.01 0.01 0.02 0.01 0.01 0.01 0.00 0.09 0.02 0.03 2.0 0.10 0.16 2.0 0.10 0.29 2.0 0.10 0.41 2.0 0.10 0.13 2.0 0.10 0.22 2.0 0.10 0.20 2.0 0.10 0.00 2.0 0.10 1.85 2.0 0.10 0.41 2.0 0.10 0.64 50 Drift 1.50 1.48 99% 51 Wash 52 F54089-8 53 F54106-8 54 F54080-8 55 F54092-8 56 F54109-8 57 F54114-8 58 F54076-8 59 F54082-8 60 F54111-8 61 F54113-8 0.00 0.04 0.05 0.04 0.04 0.03 0.04 0.06 0.09 0.06 0.07 2.0 0.10 0.75 2.0 0.10 1.05 2.0 0.10 0.73 2.0 0.10 0.87 2.0 0.10 0.67 2.0 0.10 0.80 2.0 0.10 1.17 2.0 0.10 1.75 2.0 0.10 1.19 2.0 0.10 1.40 62 Drift 1.50 1.51 101% 63 Wash 64 F54079-8 65 F54084-8 66 F54098-8 67 F54116-8 0.00 0.07 0.07 0.08 0.23 2.0 0.10 1.36 2.0 0.10 1.44 2.0 0.10 1.58 2.0 0.10 4.56 Page 2 HWI-1 59B.XLS 68 Ddft 1.50 1.51 101% 69 Wash 0.00 Page 3 HWI-1 59C.XLS 1995-06-13 13:25 OutPut of :95050BAl Operator : DDW Date of the Analysis 1995-05-08 07:30 Analysis FileName C:\SKALAR\DATAkHWIDATA\SERUM\95050BAl 1 Tracer 1.5 1.45 96% 2 Drift 1.5 1.47 98% 3 Wash 0.00 4 Standard 1 0.015 0.014 93% 5 Standard 2 0.03 0.03 104% 6 Standard 3 0.06 0.06 100% 7 Standard 4 0.09 0.09 104% 8 Standard 5 0.12 0.12 96% 9 Standard 6 0.15 0.15 103% 10 Standard 7 0.3 0.29 96% 11 Standard 8 0.6 0.61 101% 12 Standard 9 1.2 1.24 103% 13 Standard 10 1.5 1.46 98% 14 Drift 1.50 1.47 98% 15 Wash 0.00 16 Blk 1 0.03 17 Blk 2 0.01 18 Spk 1 0.08 19 Spk 2 0.09 20 F54075-12 0.03 21 F54081-12 0.02 22 F54105-12 0.03 23 F54107-12 0.02 2.0 0.10 0.60 2.0 0.10 0.26 2.0 0.10 1.66 0.004 63.00 0 2.0 0.10 1.77 0.004 63.00 0 2.0 0.10 0.63 2.0 0.10 0.43 2.0 0.10 0.60 2.0 0.10 0.40 24 F54074-12 0.02 25 F54085-12 0.01 2.0 0.10 0.42 2.0 0.10 0.29 26 Drift 1.50 1.45 97% 27 Wash 0.00 28 F54086-12 0.04 29 F54104-12 0.04 30 F54077-12 0.06 31 F54083-12 0.05 2.0 0.10 0.70 2.0 0.10 0.81 2.0 0.10 1.22 2.0 0.10 0.98 Page 1 HWI-1 59C.XLS 32 F54094-12 0.05 33 F54100-12 0.03 34 F54091-12 0.04 35 F54097-12 0.03 36 F54102-12 0.02 37 F54108-12 0.01 38 Drift 1.50 1.47 98% 39 Wash 0.00 40 F54077-12 0.02 41 F54071-12 0.00 42 F54093-12 0.00 43 F54095-12 0.00 44 F54101-12 0.00 45 F54110-12 0.00 46 F54096-12 0.00 47 F54090-12 0.02 48 F54078-12 0.02 49 F54088-12 0.04 50 Ddft 1.50 1.46 97% 51 Wash 0.00 52 F54106-12 0.05 53 F54089-12 0.03 54 F54087-12 0.03 55 F54080-12 0.03 56 F54092-12 0.02 57 F54109-12 0.03 58 F54114-12 0.04 59 F54076-12 0.03 60 F54113-12 0.03 61 F54082-12 0.05 62 D(ift 1.50 1.44 96% 63 Wash 0.00 64 F54111-12 0.07 65 F54098-12 0.07 66 F54084-12 0.07 67 F54079-12 0.06 2.0 0.10 1.03 2.0 0.10 0.62 2.0 0.10 0.90 2.0 0.10 0.67 2.0 0.10 0.46 2.0 0.10 0.20 2.0 0.10 0.39 2.0 0.10 0.00 2.0 0.10 0.00 2.0 0.10 0.00 2-0 0.10 0.00 2.0 0.10 0.00 2,0 0.10 0.04 2.0 0.10 0.49 2.0 0.10 0.32 2.0 0.10 0.79 2.0 0.10 0.96 2.0 0.10 0.65 2.0 0.10 0.64 2.0 0.10 0.53 2.0 0.10 0.44 2.0 0.10 0.64 2.0 0.10 0.72 2.0 0.10 0.68 2.0 0.10 0.66 2.0 0.10 0.95 2.0 0.10 1.40 2.0 0.10 1.39 2.0 0.10 1.30 2,0 0.10 1.25 Page 2 HWI-1 59C.XLS 68 F54116-12 0.07 2.0 0.10 1.32 69 Drift 1.50 1.52 101% 70 Wash 0.00 11" II--%Oi -S . kwoi ,;-A r- Page 3 HWI-159D.XLS 1995-06-1310:27 OutPut of:950509Al Operator :DDW Date ofthe Analysis:1995-05-0909:48 AnalysisFileName C:\SKALAR\IDATAMMIDATA\SERUM\950509Al VA 1,4 I Tracer 1.5 1.47 98% 2 Drift 1.5 1.50 100% 3 Wash 0.00 4 Standard 1 0.015 0,014 94% 5 Standard1 0.03 0.03 106% 6 Standard3 0.06 0.06 98% 7 Standard4 0.09 0.09 101% 8 Standard5 0.12 0.12 99% 9 Standard6 0.15 0.15 102% 10 Standard7 0.3 0.29 97% 11 Standard8 0.6 0.61 101% 12 Standard9 1.2 1.23 103% 13 Standard 10 1.5 1.47 98% 14 Drift 1.5 1.55 103% 15 Wash 0.00 16 Serum blk 1 0,05 17 Serum blk 2 0.03 18 Serum spk 1 0.04 19 Serum spk 2 0.05 20 Serum spk 3 0.06 21 Serum spk 4 0.07 22 Serum spk 5 0.10 23 Serum spk 6 0.07 24 Serum spk 7 0.06 25 Serwn spk 8 0.07 2.0 0.10 0.90 2.0 0.10 0.62 2.0 0.10 0.87 0.004 63.00 0.15 2.0 0.10 0.91 0.004 63.00 0.15 2.0 0.10 1.17 0.004 63.00 0.15 2.0 0.10 1.30 0.004 63.00 0.15 2.0 0.10 2.03 0.004 63.00 0.15 2.0 0.10 1.43 0.004 63.00 0.15 2.0 0.10 1.27 0.004 63.00 0.15 2.0 0.10 1.46 0.004 63.00 0.15 26 Drift 1.50 1.51 101% 27 Wash 28 F54105-24 29 F54075-24 30 F54081-24 31 F54107-24 0.00, 0.05 0.02 0.02 0.02 2.0 0.10 1.08 2.0 0.10 0.47 2.0 0.10 0.40 2.0 0.10 0.31 ILI Page 1 HWI-1 59D.XLS 32 F54104-24 33 F54086-24 34 F54074-24 35 F54085-24 36 F54083-24 37 F54100-24 0.02 0.02 0.04 0.00 0.00 0.00 2.0 0.10 0.47 2.0 0.10 0.35 2.0 0.10 0.70 2.0 0.10 0.00 2.0 0.10 0.00 2.0 0.10 0.00 38 Drift 1.50 1.47 98% 39 Wash 0.00 40 Blk 1 0.03 2.0 0.10 0.62 41 Blk 2 0.01 2.0 0.10 0.27 42 Spk 1 0.04 2.0 0.10 0.80 0.004 63..00 0.15 43 Spk 2 0.06 2.0 0.10 1.11 0.004 63 00 0.15 44 Spk 3 0.06 2.0 0.10 1.16 0.004 63.00 0.15 45 Spk 4 0.06 2.0 0.10 1.18 0.004 63.00 0.15 46 Spk 5 0.07 2.0 0.10 1.35 0.004 63.00 0.15 47 Spk 6 0.07 2.0 0.10 1.32 0.004 63.00 0.15 48 Blk 3 0.04 2.0 0.10 0.82 49 F54085-24 0.02 2.0 0.10 0.31 50 Drift 1.50 1.51 100% 51 Wash 0.00 52 F54083-24 0.02 2.0 0.10 0.42 53 F54100-24 0.02 2.0 0.10 0.31 54 F54077-24 0.02 2.0 0.10 0.34 55 F54094-24 0.01 2.0 0.10 0.27 56 F54091-24 0.01 2.0 0.10 0.28 57 F54108-24 0.01 2.0 0.10 0.27 58 F54102-24 0.01 2.0 0.10 0.18 59 F54097-24 0.02 2.0 0.10 0.48 60 F54071-24 0.01 2.0 0.10 0.24 61 F54093-24 0.01 2.0 0.10 0.21 62 Drift 1.50 1.49 100% 63 Wash 0.00 64 F54095-24 0.02 2.0 0.10 0.42 65 F54101-24 0.02 2.0 0.10 0.34 66 F54110-24 0.02 2.0 0.10 0.30 67 F54096-24 0.01 2.0 0.10 0.23 Page 2 HWI-I 59D.XLS ;b. 1,Lc 68 69 70 71 @7 12 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 I 2 3 4 5 6 7 8 F54090-24 F54078-24 F54088-24 F54106-24 F54089-24 F54087-24 Drift Wash F54080-24 F54092-24 F54109-24 F54114-24 Blk 1 Btk 2 Spk 1 Spk 2 F54113-24 F54082-24 Drift Wash F54111-24 F54098-24 F54084-24 F54079-24 F54116-24 Drift Wash Tracer Drift Wash F54106-24 F54089-24 F54087-24 Drift Wash 0.05 0.03 0.02 0.81 0.44 0.57 1.50 0.22 15% 0.00 0.06 0.05 0.04 0.03 0.02 0.01 0.09 0.11 0.06 0.04 1.50 1.42 95% 0.00 0.06 0.05 0.05 0.05 0.06 1.50 1.46 97% 0.00 1.50 1.50 1.51 101% 1.52 101% 0.00 0.04 0.05 0.04 1.50 1.62 108% 0.00 2.0 0.10 1.02 2.0 0.10 0.52 2.0 0.10 0.48 2-0 0.10 16.10 2.0 0.10 8.70 2.0 0.10 11.48 2.0 0.10 1.21 2.0 0.10 1.02 2.0 0.10 0.78 2.0 0.10 0.64 2.0 0.10 0.41 2.0 0.10 0.29 2.0 0.10 1.75 0.004 63.00 0.15 2.0 0.10 2.13 0.004 63.00 0.15 2.0 0.10 1.17 2.0 0.10 0.86 2.0 0.10 1.18 2.0 0.10 1.10 2.0 0.10 1.03 2.0 0.10 1.00 2.0 0.10 1.20 2.0 0.10 0.88 2.0 0.10 0.99 2.0 0.10 0.81 Page 3 HWI-1 59E.XLS 1995-06-13 10:27 OutPut of :950511 Al Operator : DDW Date ofthe Analysis 1995-05-11 08:16 Analysis FileName C:ISKALAR\DATA\HWIDATA\SERUM\950511AI I Tracer 1.50 1.47 98% 2 Drift 1.50 1.47 98% 3 Wash 0.00 4 Standard 1 0.015 0.016 109% 5 Standard 2 0.03 0.03 96% 6 Standard 3 0.06 0.06 98% 7 Standard 4 0.09 0.09 102% 8 Standard 5 0.12 0.12 100% 9 Standard 6 0.15 0.15 102% 10 Standard 7 0.30 0.29 96% 11 Standard 8 0.60 0.61 101% 12 Standard 9 1.20 1.23 103% 13 Standard 10 1.50 14 Drift 1.50 1.47 98% 1.51 101% 15 Wash 0.00 16 F54113-48 0.07 17 F54082-48 0.07 2.0 0.10 1.44 2.0 0.10 1.37 18 F54111-48 0.08 19 F54098-48 0.07 20 F54084-48 0.09 21 F54079-48 0.05 22 F54116-48 0.08 23 F54113-192 0.08 2.0 0.10 1.52 2.0 0.10 1.37 2.0 0.10 1.73 2.0 0.10 1.05 2,0 0.10 1.69 2.0 0.10 1.55 24 F54082-192 0.06 2.0 0.10 1.28 25 F54111-192 0.05 2.0 0.10 1.02 26 Drift 1.50 1.44 96% 27 Wash 0.00 28 F54098-192 0.06 2.0 0.10 1.25 29 F54079-192 30 F54084-192 0.06 0.07 2.0 0.10 1.28 2.0 0.10 1.46 31 F54116-192 0.06 2.0 0.10 1.15 Page I HWI-1 59E.XLS 32 F54113-360 33 F54082-360 34 F54111-360 35 F54098-360 36 F54079-360 37 F54084-360 0.06 0.04 0.06 0.05 0.07 0.07 2.0 0.10 1.17 2.0 0.10 0.71 2.0 0.10 1.22 2.0 0.10 1.04 2.0 0.10 1.36 2.0 0.10 1.40 38 Drift 1.50 1.48 99% 39 Wash 40 F54116-360 41 BLK 1 42 SPK 1 43 SPK 2 44 F54082-D22 45 F54111-D22 46 F54079-D22 47 F54116-D22 48 F54082-D28 49 F54111-D28 0.00 0.10 0.02 0.07 0.10 0.06 0.06 0.05 0.05 0.06 0.04 2.0 0.10 1.92 2.0 0.10 0.38 2.0 0.10 1.45 0.004 63,00 0.1 2.0 0.10 2.00 0.004 63.00 0. 2.0 0.10 1.17 2.0 0.10 1.14 2.0 0.10 1.07 2.0 0.10 1.08 2.0 0.10 1.21 2.0 0.10 0.89 50 Drift 1.50 1.52 101% 51 Wash 0.00 52 F54079-D28 0.04 2.0 0.10 0.86 53 F54116-D28 0.05 2.0 0.10 1.00 54 F54099-4 0.03 2.0 0.10 0.63 55 F54099-8 0.03 2.0 0.10 0.60 56 F54099-12 0.09 2.0 0.10 1.79 57 F54099-24 0.11 2.0 0.10 2.15 58 F54099-48 0.06 2.0 0.10 1.14 59 F54099-i92 0.04 2.0 0.10 0.74 60 F54099-360 0.03 2.0 0,10 0.61 61 F54 2.0 o.io 62 Drift 1.50 1.50 100% 63 Wash 0.00 64 F54112-4 65 F54112-8 66 F54112-12 67 F54112-24 0.05 0.07 0.08 0.06 2.0 0.10 1.05 2.0 0.10 1.47 2.0 0.10 1.65 2.0 0.10 1.25 Page 2 HWI-1 59E.XLS 68 F54112-48 0.08 69 F54112-192 0.07 2.0 0.10 1.63 2.0 0.10 1.45 70 F54112-360 71 SPK 63-1 72 SPK 63-2 73 SPK 63-3 0.05 0.08 0.10 0.07 2.0 0.10 0.90 2.0 0.10 1.50 0.004 63.00 0.1 2.0 0.10 1.95 0.004 63.00 0. 2.0 0.10 1.38 0.004 63.00 0. 74 Ddft 1.50 1.47 98% 75 Wash 76 SPK 126-1 77 SPK 126-2 78 SPK 126-3 0.00 0.11 0.15 0.15 2.0 0.10 2.12 0.004 126.00 0. 2.0 0.10 2.90 0.004 126.00 0. 2.0 0.10 3.07 0.004 126.00 0. 79 Drift 1.50 1.50 100% 80 Wash 0.00 Page 3 HWI-1 59F.XLS 1995-06-1312:41 OutPut of :950524AI Operator DDW Date ofthe Analysis: 1995-05-24 11:39 AnalysisFileName C:\SKALAR\DATA\HWIDATA\SERUM\950524Al I Tracer 1.5 1.45 97% 2 Drift 1.5 1.47 98% 3 Wash 0.00 4 Standard 1 0.015 0.018 120% 5 Standard2 0.03 0.03 92% 6 Standard 3 0.06 0.06 95% 7 Standard4 0.09 0.09 105% 8 Standard5 0.12 0.12 99% 9 Standard6 0.15 0.16 103% 10 Standard7 0.3 0.28 95% 11 Standard8 0.6 0.61 102% 12 Standard9 1.2 1.24 104% 13 Standard 10 14 Drift 1.5 1.50 1.46 97% 1.44 96% 15 Wash 16, BLK 1 17 BLK 2 18 SPK 63-1 19 SPK 63-2 20 SPK 63-3 21 SPK 63-4 22 SPK 63-5 23 SPK 63-6 24 SPK 63-7 25 BLK 3 0.00 0.03 0.02 0.08 0.08 0.08 0.07 0.09 0.08 0.08 0.03 2.0 0.10 0.66 2.0 0.10 0.34 2.0 0.10 1.69 0.004 63.00 0. 2.0 0.10 1.52 0.004 63.00 0. 2.0 0.10 1.54 0.004 63.00 0. 2.0 0.10 1.37 0.004 63.00 0. 2.0 0.10 1.72 0.004 63.00 0. 2.0 0.10 1.66 0.004 63.00 0. 2.0 0.10 1.64 0.004 63.00 0. 2.0 0.10 0.53 26 Drift 1.50 1.46 97% 27 Wash 28 BLK 4 0.00 0.03 2.0 0.10 0.55 Page I HWI-1 59F.XLS 29 F54088-48 0.02 2.0 0.10 0.31 30 F54099-48 0.02 2.0 0.10 0.47 31 F54089-48 0.01 2.0 0.10 0.22 32 F54087-48 0.02 2.0 0.10 0.38 33 F5408048 0.01 2.0 0.10 0.28 34 F54092-48 0.01 2.0 0.10 0.19 35 F5410948 0.02 2.0 0.10 0.43 36 F5411448 0,02 37 F54088-DY8 0.02 2.0 0.10 0.46 2.0 0.10 0.32 38 Drift 1.50 1.49 99% 39 Wash 0.00 40 F54099-DY8 0.02 41 SPK 63-8 0.08 42 SPK 63-9 0.08 43 SPK 63-10 0.08 2.0 0.10 0.34 2.0 0.10 1.62 0.004 63.00 0. 2.0 0.10 1.55 0.004 63.00 0. 2.0 0.10 1.58 0.004 63.00 0. 44 BLK 1 0.03 2.0 0.10 0.55 45 BLK 2 0.01 2.0 0.10 0.17 46 SPK 63-1 0.06 2.0 0.10 1.19 0.004 63.00 0. 47 SPK 63-2 0.08 2.0 0.10 1.53 0.004 63.00 0. 48 SPK 63-3 0.08 2.0 0.10 1.68 0.004 63.00 0. 49 F54089-DY8 0.02 2.0 0.10 0.49 50 Drift 1.50 1.47 98% 51 Wash 0.00 52 F54087-DY8 0.03 53 F54080-DY8 0.03 54 F54092-DY8 0.04 55 F54109-DY8 0.03 56 F54114-DY8 0.04 57 F54088-DI5 0.00 58 F54099-DI5 0.03 59 F54089-DI5 0.02 60 F54087-DI5 0.02 61 F54080-DI5 0.01 2.0 0,10 0.66 2.0 0.10 0.65 2.0 0.10 0.73 2.0 0.10 0.56 2.0 0.10 0.73 2.0 0.10 0.00 2.0 0.10 0.53 2.0 0.10 0.33 2.0 0.10 0.35 2.0 0.10 0.21 Page 2 HWI-1 59F.XLS 62 Drift 1.50 1.47 98% 63 Wash 0.00 64 F54092-DI5 0.02 2.0 0.10 0.43 65 F54109-D 15 0.02 2.0 0.10 0.50 66 F54114-DI5 0.01 2.0 0.10 0.29 67 F54089-D22 0.02 2.0 0.10 0.32 68 F54087-D22 0.01 2.0 0.10 0.27 69 F54109-D22 0.01 2.0 0.10 0.29 70 F54114-D22 0.02 2.0 0.10 0.34 71 F54089-D28 0.01 2.0 0.10 0.23 72 F54087-D28 0.02 2.0 0.10 0.34 73 F54109-D28 0.02 2.0 0.10 0.34 74 Drift 1.50 1.46 97% 75 Wash 0.00 76 F54114-D28 0.02 77 SPK 63-4 0.04 78 SPK 63-5 0.06 79 SPK 63-6 0.08 80 SPK 63-7 0.08 81 F54105-48HR 0.02 82 F54075-48HR 0.03 2.0 0.10 0.36 2.0 0.10 0.76 0.004 63.00 0. 2.0 0.10 1.22 0.004 63.00 0. 2.0 0.10 1.53 0.004 63.00 0. 2.0 0.10 1.50 0.004 63.00 0. 2.0 0.10 0.36 2.0 0.10 .0.52 83 Drift 1.50 1.41 94% 94 Wash 0.00 Page 3 .995-05-04 16:44 output of 950504Al ,oftware version 6.1 cl990,93 'perator : DDW )ate of the Analysis : 1995-05-04 08:18 ,.nalysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950504Al AIL%'O (032 Is ck 7luoride 1.5 :alibration order = Inverse Logarithm IDlope s x - cl Zesult = 10L s x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode -i2= il = to = -0.00000 0.00063 -1.24458 Fluoride L @alibration order = 2 7orrelation lesult = a2 r = 0.99928 X2 + al * X + aO i12 = il = -10 = 0.00000 0.00018 -0.00141 'ampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SA1000 :1 : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. Diluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample Dilution runs User file : TXT Reproces : No 995-05-04 1.6:44 OutPut of 950504Al 'luoride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 : Debubbled : Yes :0 : No : No : 4095 :0 : Off sl standard : Ignore s2 standard : Ignore s3 standard : Ignore s4 standard : Ignore s5 standard : Ignore s6 standard : s7 standard : s8 standard : 0.150 0.300 0.600 S9 standard : slO standard : 1.200 1.500 order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape start ignore : Pointed : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula Output @'luoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off '.995-05-04 16:44 OutPut of : 950504Al sl standard : 0.015 s2 standard : 0.030 s3 standard : 0.060 s4 standard : 0.090 s5 standard : 0.120 s6 standard : 0.150 s7 standard : Ignore s8 standard : Ignore S9 standard : Ignore slO standard : Ignore order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sac Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter No Regeneration No formula c4:=c3 output .995-05-04 16:44 output of : 950504Al Fluoride 1.5 Fluoride L PPM PPM los Typ Ident Ch Result F Time ;t iw t d w sl i s2 i s3 7 s4 s5 s6 -0 s7 -.L S8 -2 s9 -3 slO -.4 d -5 w .-.6 u .7 u -8 u .-9 u '0 u 11 u '-2 u -3 u .11-4 u -5 u -@6 d -@.7 w -'B u u 30 u 31 u 32 u -;3 u --4 u -3 u 26 u 'j7 u 38 d '-9 w u u 2u 43 u A:4 u 45 u 46 u 47 u 48 u -,.9 u 50 d 51 w 52 u r3 U Initial Wash 3 Tracer 3 Drift 3 Wash 3 Standard 1 3 Standard 2 3 Standard 3 3 Standard 4 3 Standard 5 3 Standard 6 3 Standard 7 3 Standard 8 3 Standard 9 3 Standard 10 3 Drift 3 Wash 3 Blk 1 3 Blk 2 3 Spk 63-1 3 Spk 63-2 3 Spk 94.5-1 3 Spk 94.5-2 3 Spk 126-1 3 Spk 126-2 3 Spk 126-3 3 Spk 126-4 3 Drift 3 Wash 3 F54081-4 3 F54075-4 3 F54107-4 3 F54105-4 3 F54074-4 3 Blk-I 3 Blk-2 3 Spk 63-1 3 Spk 63-2 3 Spk 63-3 3 Drift 3 Wash 3 Spk 126-1 3 Spk 126-2 3 Spk 126-3 3 Spk 126-4 3 Spk 126-5 3 Spk 126-6 3 Spk 126-7 3 F54085-4 3 F54080-4 3 F54104-4 3 Drift 3 Wash 3 F54077-4 3 F54083-4 3 0.057 1.462 1.467 0.057 0.065 0.073 0.090 0.109 0.132 0.153 0.288 0.609 1.226 1.474 1.472 0.057 0.077 0.070 0.095 0.098 0.098 0.080 0.171 0.112 0.142 0.149 1.425 0.057 0.079 0.073 0.069 0.066 0.070 0.828 0.402 0.100 0.111 0.113 1.464 0.057 0.146 0.147 0.144 0.151 0.138 0.157 0.129 0.083 0.072 0.072 1.467 0.057 0.071 0.066 65 212 387 629 740 915 1087 1263 1436 1611 1787 1961 2137 2313 2487 '2729 2838 3013 3188 3364 3538 3714 3888 4062 4238 4414 4588 4756 4938 5113 5291 5465 5641 5815 5989 6165 6339 6515 6689 6911 7040 7215 7390 7566 7740 7916 8092 8266 8442 8623 8790 9032 9138 9314 Ch Result F Time 4 #.#### 0 4 1.0733 0 4 1.0775 0 4 0 4 0.0150 0 4 0.0304 0 4 0.0594 0 4 0.0891 0 4 0.1221 0 4 0.1490 0 4 0.2865 0 4 0.5232 0 4 0.9080 0 4 1.0831 0 4 1.0810 0 4 0 4 0.0385 0 4 0.0242 0 4 0.0686 0 4 0.0736 0 4 0.0732 0 4 0.0433 0 4 0.1698 0 4 0.0937 0 4 0.1343 0 4 0.1431 0 4 1.0452 0 4 0 4 0.0420 0 4 0.0306 0 4 0.0229 0 4 0.0172 0 4 0.0255 0 4 0.6608 0 4 0.3788 0 4 0.0767 0 4 0.0929 0 4 0.0963 0 4 1.0750 0 4 0 4 0.1397" 0 4 0.1417 0 4 0.1368 0 4 0.1467 0 4 0.1292 0 4 0.1533 0 4 0,1179 0 4 0.0492 0 4 0.0278 0 4 0.0291 0 4 1.0775 0 4 #.#### 0 4 0.0265 0 4 0.0163 0 Page 1 of 2 e -L5 .995-05-04 16:44 output of : 950504Al Fluoride 1.5 Fluoride L PPM PPM )os Typ Ident Ch Result F Time Ch Result F Time ;14 u F54094-4 3 0.067 9488 4 0.0181 0 5u F54100-4 3 0.067 9666 4 0.0196 0 . 16 u F54091-4 3 0.067 9842 4 0.0181 0 -)7 u Elk 1 -3 0.069 10016 4 0.0220 0 58 u Elk 2 3 0.064 10191 4 0.0130 0 59 u Spk 1 3 0.103 10367 4 0.0800 0 ')0 u Spk 2 3 0.105 10543 4 0.0835 0 )l u F54097-4 3 0.067 10717 4 0.0200 0 ';2 d Drift 3 1.436 10891 4 1.0537 0 -3 w wash 3 0.057 11066 4 0 @4 u F54102-4 3 0.067 11241 4 0.0192 0 u F54092-4 3 0.064 11419 4 0.0141 0 -16 u F54080-4 3 0.072 11591 4 0.0287 0 17 u F54106-4 3 0.075 11769 4 0.0340 0 ')8 u .I- pxjVo.&4--4- 3 0.067 11942 4 0.0181 0 -)9 u F54/088-4 3 0.067 12116 4 0.0200 0 70 U F54,YO87-4 3 0.070 12291 4 0.0248 0 71 U F54XllO-4 3 0.064 12462 4 0.0123 0 '2 u F54096-4 3 0.061 12642 4 0.0064 0 u F54090-4 3 0.064 12818 4 0.0138 0 -4 d Drift 3 1.455 12992 4 1.0681 0 5w Wash 3 0.057 13159 4 0 '6 u F54078-4 3 0.067 13342 4 0.0194 0 -.17 u F54101-4 3 0.061 13516 4 0.0076 0 78 u F54095-4 3 0.063 13692 4 0.0120. 0 79 u F54093-4 3 0.068 13868 4 0.0213 0 30 u F54071-4 3 0.063 14042 4 0.0118 0 31 u F54108-4 3 0.062 14213 4 0.0102 0 32 u F54116-4 3 0.139 14393 4 0.1307 0 33 u F54098-4 3 0.091 14568 4 0.0624 0 34 u F54084-4 3 0.108 14744 4 0.0889 0 35 u F54079-4 3 0.152 14919 4 0.1471 0 -"6 d Drift 3 1.478 15093 4 1.0857 0 @"7 w Wash 3 0.057 15332 4 0 7,8 u F54113-4 3 0.124 15444 4 0.1114 0 -? u F54111-4 3 0.131 15619 4 0.1210 0 30 u F54082-4 3 0.096 15795 4 0.0702 0 91 u F54076-4 3 0.082 15967 4 0.0460 0 92 u F54114-4 3 0.080 16145 4 0.0431 0 93 u F54109-4 3 0.065 16319 4 0.0160 0 94 d Drift 3 1.468 16493 4 1.0780' 0 95 w Wash 3 0.057 16650 4 0 -dt rw RunOut Wash 3 0.057 16968 4 0 Page 2 of 2 oool(31'@i Cal;Lbra,!L,i.ceunrve of 9'-@OS04AI Fluoride CiCi' c 00 Li@j Ieasured 90 Cali-hra-3-iorctf,rve egi:'4qOSO4A! Fluoridp 1.4 ;4279 jL 91 "3r UD-C Measured a r i h:t 0()01,-1*0 Raw dat4-o2f- 9E-zOS04A! Fluoride 1.5 Cl Cdo f@ ,", III -45 2.5 I. t .19 E riea-'s 000171 OMER, --:atao'@ rSO504L.- Flucride 1.5 5-k 4JO -4-L Lis J'l ki IL Time ct L-x'-;Lt F H a p t,r 21-'.1- P rrd-- "I Fje a i<s looo I sc @'U'4b -1 Raw dat.-=of Be icli!oride1 .5 777 64 I rev lo T ime 1 Crtl-'r'=-Pd'PLetaks 1 -?Co -ti 137601 F;aw dal-a of 85&rjS04A Fluoride 1-S 4 go t qo ca T 7 .sc= x.@ F4=HeIF3 Crti-P=@dit 000174 995-05-04 16:48 OutPut of : 950504Bl @oftware version 6.1 cl990,93 perator : DDW ate of the Analysis : 1995-05-04 13:07 ,nalysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950504Bl 'luoride 1.5 .'alibrationorder = Inverse Logarithm 31ope s x - cl "esult = 10[ s x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode ,2 = il = 10 = -0.00000 0.00062 - -1.24651 7luoride L -'alibrationorder = 2 7orrelation r = 0.99962 @iesult= a2 X2 + al * x + aO ,2 = il = -10 = 0.00000 0.00020 -0.00460 3ampler Type Number Sample Time Wash Time Air Time' Take up special needle Height : SAIOOO :1 : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. )iluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample :1 Dilution runs : 1 User file : TXT Reproces, : No 42bcis.l oool7ri ?3 .995-05-04 1.6:48 output of 950504Bl Iluoride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 : Debubbled : Yes :0 : No : No : 4095 :0 : Off sl standard : s2 standard : s3 standard : s4 standard : s5 standard : s6 standard : s7 standard : S8 standard : S9 standard : slO standard : Order : Inverse Dimension : PPM start Value trigger Limit Peak shape start ignore eNd ignore Measure window Filter Regeneration formula output Ignore Ignore Ignore Ignore Ignore 0.150 0.300 0.600 1.200 1.500 Logarithm : 500 DU : 1800 Sec : Pointed : 60 Sec : 120 Sec : 75 % No No -@'luoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off 00017C) .995-05-04 16:48 Output of sl standard : 0.015 s2 standard : 0.030 s3 standard : 0.060 S4 standard : 0.090 s5 standard : s6 standard : 0.120 0.150 s7 standard : Ignore S8 standard : Ignore S9 standard ; Ignore slO standard : Ignore order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape start ignore : Pointed : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula c4:=c3 output 950504Bl 00017,"r .995-05-04 16:48 Output of 950504Bl Fluoride 1.5 Fluoride L )os Typ Ident ppm Ch Result F Time ppm Ch Result F Time It iw Initial Wash 3 0.057 65 4 #.#### 0 t Tracer 3 1.467 212 4 0.8037 0 d Drift 3 1.473 388 4 0.8066 0 w Wash 3 0.057 629 4 0 sl standard 1 3 0-065 740 4 0.0140 0 s2 Standard 2 3 0-073 910 4 0.0311 0 s3 Standard 3 3 0.089 1089 4 0.0615 0 s4 standard 4 3 0.105 1264 4 0.0877 0 s5 standard 5 3 0.128 1440 @4 0.1206 0 s6 standard 6 3 0-153 1614 4 0.1501 0 0 s7 standard 7 3 0-291 1790 4 0.2729 0 S8 Standard 8 3 0.605 1964 4 0.4486 0 . -2 s9 Standard 9 3 1.228 2138 4 0.7030 0 -3 slo Standard 10 3 1.475 2314 4 0.8073 0 -4 d Drift 3 1.448 2488 4 0.7951 0 -5 w Wash 3 0.057 2709 4 #.#### 0 -6 u F54075-8 3 0.073 2837 4 0.0319 0 -7 u F54081-8 3 0.067 3013 4 0.0199 0 .8 u F54105-8 3 0.071 3186 4 0.0274 0 -9 u F54107-8 3 0.065 3361 4 0.0154 0 @o u F54074-8 3 0.066 3541 4 0.0176 0 21 u F54085-8 3 0.073 3712 4 0.0311 0 22 u F54086-8 3 0.075 3891 4 0.0357 0 23 u BLK-1 3 0.068 4067 4 0.0211 0 24 u BLK-2 3 0.092 4241 4 0.0663 0 25 u SPK 1 3 0.113 4415 4 0.1002 0 26 d Drift 3 1.439 4589 4 0.7912 0 27 w Wash 3 0.057 4828 4 #.#### 0 '8 u SPK 2 3 0.098 4941 4 0.0774 0 29 u F54104-8 3 0.075 5112 4 0.0366 0 -10 u F54077-8 3 0.072 5301 4 0.0292 0 u F54083-8 3 0.068 5461 4 0.0217 0 32 u F54094-8 3 0-069 5642 4 0.0227 0 33 u F54100-8 3 0.067 5813 4 0.0203 0 34 u F54091-8 3 0.061 5987 4 0.0061 0 u F54097-8 3 0.060 6169 4 0.0027 0 36 u F54102-8 3 0.065 6339 4 0.0146 0 J7 u F54108-8 3 0.059 6513 4 0.0004 0 38 d Drift 3 1.397 6690 4 0.7727 0 39 w Wash 3 0.057 6854 4 #.#### 0 40 u F54071-8 3 0.062 7040 4 0.0081 0 41 u F54093-8 3 0.065 7214 4 0.0144 0 42 u F54095-8 3 0.067 7392 4 0.0203 0 43 u F54101-8 3 0.061 7562 4 0.0063 0 44 u F54078-8 3 0.063 7740 4 0.0108 0 45 u F54090-8 3 0-063 7916 4 0.0101 0 46 u F54096-8 3 0-058 8088 4 #.#### 0 47 u F54110-8 3 0.108 8266 4 0.0923 0 48 u F54087-8 3 0.068 8441 4 0.0207 0 49 u F54088-8 3 0.073 8615 4 0.0321 0 50 d Drift 3 1.481 8790 4 0.8103 0 51 w Wash 3 0.057 8938 4 #.#### 0 52 u F54089-8 3 0.076 9141 4 0.0376 0 "I u F54106-8 3 0.084 9315 4 0.0525 0 Page 1 of 2 00017,9 .995-05-04 16:48 output of : 950504Bl Fluoride 1.5 Fluoride L ppm PPM os Typ Ident Ch Result F Time Ch Result F Time 4u F54080-8 3 0.075 9491 4 0.0364 0 5u F54092-8 3 0.079 9667 4 0.0433 0 6u F54109-8 3 0.074 9841 4 0.0333 0 7u F54114-8 3 0.077 '10015 4 0.0402 0 8u F54076-8 3 0.087 10193 4 0.0585 0 9u F54082-8 3 0.105 10367 4 0.0877 0 0u F54111-8 3 0.088 10543 4 0.0593 0 1u F54113-8 3 0.094 10717 4 0.0700 0 2d Drift 3 1.512 10891 4 0.8247 0 @.3 w Wash 3 0.057 11093 4 #.#### 0 @4 u F54079-8 3 0.093 11240 4 0.0681 0 iu F54084-8 3 0.095 11416 4 0.0718 0 6u F54098-8 3 0.099 11591 4 0.0792 0 7u F54116-8 3 0.228 11766 4 0.2236 0 8d @9 w Drift 3 1.512 11941 4 0.8245 0 Wash 3 0.057 12097 4 0 4*t rw RunOut Wash 3 0.057 12416 4 0 Page 2 of 2 oOO17,@1) Cali..-iraticounrve of SSOSCABI Fluoride L . -6 i -C, Hea-r-e@Lu4 r 0(.)01-80 513 Calibration c3jrve o-,;9750SO481 Fluoride i.S /T i / rv u 0 Measured 409S 0()0181 Saw data of 950SO4 64 !7r4 Fluoride 1--q -d ftmi!:ZA 10 1 Ilk 0 T !::4A=Help Cr4'1-P=Edit Peaks 1! 51'Cio oools;4 Raw data of 950504BI F'Luoride 1.5 94 ?!36 'All 40 4-S @j T i-te x i FI=Help Crtl-r"=Edi%, peaii%,sil 0()01810"1 L4. Raw data of 850504BI Fluoride 1.5 cz4 fi i cl, el 9 11137-!=HeIP to 1 .,!too fI (do;o Ir Time r .3L Peak:5 000184 -995-05-08 12:46 1@,oftware version 6.1 output of cl990,93 950508Al )perator : DDW )ate of the Analysis : 1995-05-08 07:30 knalysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950508AI T 4 -LO'%S. J (..'Z3cl -15'4 ?luoride 1.5 -alibration order = Inverse Logarithm 31ope s x - cl Result = 101 s I x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode 12 = :il = .10 = -0.00000 0.00066 -1.26704 Fluoride L ,alibration order 2 @!orrelation r = 0.99873 Result a2 X2 + al * x + aO A2 = il = -0.00000 0.00021 -0.00833 3ampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SAIOOO :1 : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. Diluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample :1 Dilution runs : 1 U;er file : TXT Reproces : No .995-05-08 12:46 output of 'luoride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 : Debubbled : Yes :0 : No : No : 4095 :0 : Off sl standard : Ignore s2 standard : Ignore s3 standard : Ignore s4 standard : Ignore s5 standard : Ignore s6 standard : 0.150 s7 - standard : 0.300 S8 standard : 0.600 S9 standard : 1.200 slO STandard : 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula output ?luoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off 950508AI ooolh,(; -995-05-08 12:46 output Of sl standard : 0.015 s2 standard : 0.030 s3 standard : 0.060 s4 standard : 0.090 s5 standard : 0.120 s6 standard : 0.150 s7 standard : Ignore S8 standard : Ignore S9 standard : Ignore slO standard : Ignore order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula c4:=c3 output 950508AI -995-05-08 12:46 OutPut of 950508AI Fluoride 1.5 Fluoride L PPM PPM los Typ Ident Ch Result F Time Ch Result P Time it iw Initial Wash 3 0.054 65 4 #.#### 0 t Tracer 3 1.447 210 4 0.7320 0 d Drift 3 1.466 384 4 0.7393 0 3w Wash 3 0.054 625 4 #.#### 0 sl Standard 1 3 0.063 734 4 0.0139 0 s2 Standard 2 3 0.071 910 4 0.0313 0 s3 Standard 3 3 0.086 1086 4 0.0597 0 s4 Standard 4 3 0.107 1260 4 0.0932 0 s5 Standard 5 3 0.124 1436 4 0.1156 0 s6 Standard 6 3 0.154 1611 4 0.1514 0 ;-0 s7 Standard 7 3 0.287 1785 4 0.2640 0 s8 Standard 8 3 0.607 1961 4 0.4282 0 @-2 s9 Standard 9 3 1.239 2134 4 0.6557 0 . -3 slO Standard 10 3 1.464 2309 4 0.7386 0 -4 d Drift 3 1.474 2485 4 0.7425 0 -5 w Wash 3 0.054 2718 4 #.#### 0 -.6 u Blk 1 3 0.071 2835 4 0.0298 0 -7 u Elk 2 3 0.063 3010 4 0.0128 0 -8 u Spk 1 3 0.100 3187 4 0.0829 0 9u Spk 2 3 0.104 3362 4 0.0883 0 .,o u F54075-12 3 0.071 3538 4 0.0315 0 ,l u F54081-12 3 0.067 3712 4 0.0217 0 12 u F54105-12 3 0.071 3886 4 0.0298 0 13 u F54107 -12 3 0.066 4057 4 0.0198 0 '.4 u F54074-12 3 0.066 4235 4 0.0209 0 @,5 u F54085-12 3 0.063 4402 4 0.0143 0 26 d Drift 3 1.454 4588 4 0.7348 0 -'7 w Wash 3 0.054 4778 4 0 .@8 u F54086-12 3 0.073 4937 4 0.0352 0 29 u F54104-12 3 0.076 5110 4 0.0403 0 30 u F54077-12 3 0.087 5286 4 0.0610 0 31 u F54083-12 3 0.081 5467 4 0.0492 0 32 u F54094-12 3 0.082 5637 4 0.0514 0 33 u F@4100-12 3 0.071 5813 4 0.0309 0 34 u F54091-12 3 0.078 5991 4 0.0448 0 u F54097-12 3 0.072 6165 4 0.0335 0 36 u F54102-12 3 0.067 6337 4 0.0232 0 37 u F54108-12 3 0.062 6515 4 0.0100 0 38 d Drift 3 1.467 6687 4 0.7399 0 :,)9 w Wash 3 0.054 6853 4 0 -:o u F54077-12 3 0.066 7037 4 0.0194' 0 'Itl u F54071-12 3 0.054 A 7212 4 0 @2 u F54093-12 3 0.054 A 7387 4 0 43 u F54095-12 3 0.054 A 7562 4 0 !4 u F54101-12 3 0.054 A 7737 4 0 45 u F54110-12 3 0.054 A 7912 4 0 -L6 u F54096-12 3 0.058 8088 4 0.0021 0 u F54090-12 3 0.068 8264 4 0.0243 0 @8 u F54078-12 3 0.064 8438 4 0.0160 0 ,@9 u F54088-12 3 0.075 8611 4 0.0397 0 50 d Drift 3 1.457 8789 4 0.7356 0 :jl w Wash 3 0.054 8995 4 #.#### 0 52 u F54106-12 3 0.080 9141 4 0.0478 0 u F54089-12 3 0.072 9315 4 0.0326 0 Page 1 of 2 oo01 ,.995-05-08 12:46 OutPut of 950508Al Fluoride 1.5 Fluoride L PPM ppm los Typ Ident Ch Result F Time Ch Result F Time ')4 u F54087-12 3 0.072 9491 4 0.0320 0 .5 u F54080-12 3 0.069 9664 4 0.0266 0 .6 u F54092-12 3 0.067 9839 4 0.0222 0 u F54109-12 3 0.072 10017 4 0.0322 0 :8 u F54114-12 3 0.074 10193 4 0.0362 0 -9 u F54076-12 3 0.073 10367 4 0.0341 0 @O u F54113-12 3 0.072 10540 4 0.0330 0 ..,l u F54082-12 3 0.080 10716 4 0.0475 0 2d Drift 3 1.442 10890 4 0.7301 0 3w Wash 3 0.054 11058 4 #.#### 0 :4 u F54111-12 3 0.092 11242 4 0.0701 0 3u F54098-12 3 0..092 11418 4 0.0697 0 -6 u F54084-12 3 0.089 11590 4 0.0650 0 -.7 u F54079-12 3 0.088 11764 4 0.0623 0 ,8 u P54116-12 3 0.090 11943 4 0.0659 0 @9 d Drift 3 1.515 12116 4 0.7597 0 -0 w Wash 3 0.054 12348 4 0 rt rw RunOut Wash 3 0.054 12591 4 0 Page 2 of 2 i.@iic-kurveoT"qSOSCl8iAlFluoride L -W j. LZ ij- Ll Measured 90c, 0001!)0 4 S 7 -2 Measul,,-ed 409C Raw da4l.-aol4 a Fluoride 1.5 4 i T4. iA 0()019" Raw Llat 1:)f i-k4ie -q also epo 3S 4SP,O i =He 1,-3 1.;r T it-e peaks Raw datp- of BSC':-)08A! F'Luoride l.i'-z t7 IL Ti 144 Crti--F=L-d,i,t peaks 1995-05-09 t5:09 output of 950509Al 3oftware version 6.1 cl990,93 )perator : DDW Date of the Analysis : 1995-05-09 09:48 knalysis File Name : C:\SKALAR\DATA\HTtlIDATA\SERUM\950509Al A^I>T 4-LO-R-5.) ?luoride 1.5 '.alibration order = Inverse Logarithm Slope s r X - cl -1 Result = 10[ - s i x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 = al = ao = -0.00000 0.00061 -1.24329 ?luoride L -alibration order = 2 Co.r.relation r = 0.99974 Result = a2 X2 + al * x + aO a2 = al = ao = 0.00000 0.00020 -0.00554 :"ampler Type Number Sample Time Wash Time Air Time Take up special needle Height SA1000 :1 : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. Diluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample :I Dilution runs :1 User file : TXT Reproces : No 00019r) '!995-05-09 15:09 output of 950509AI ?luoride 1.5 Path number Signal type Decolor system Number dilute Resample di-1 Threshold diG output Window event 3 : Debubbl@ed : Yes :0 : No : No : 4095 :0 : Off sl standard : Ignore s2 standard : Ignore s3 standard : Ignore s4 standard : Ignore s5 standard : Ignore s6 standard : 0.150 s7 standard : 0.300 s8 standard : 0.600 S9 standard : 1.200 slO standard : 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore *120 Sec Measure window 75 % Filter No Regeneration No formula output Fluoride L Path number Sianal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off L995-05-09 15:09 output of : 950509AI sl standard : 0.015 s2 standard : 0.030 s3 standard : 0.060 s4 standard : 0.090 s5 standard : 0.120 s6 standard : 0.150 s7 standard : Ignore S8 standard : Ignore S9 standard : Ignore slO standard : Ignore order : 2 Dimerision : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter No Regeneration No formula c4:=c3 output 0()019 495-05-09 15:09 OutPut of : 950509Al Fluoride 1.5 Fluoride L PPM PPM ,os Typ Ident Ch Result F Time Ch Result F Time -t iw Initial Wash 3 0.057 65 4 #.#### 0 t Tracer 3 1.474 209 4 0.7917 0 d Drift 3 1.502 384 4 0.8031 0 w Wash 3 0.057 626 4 0 sl Standard 1 3 0.065 734 4 0.0141 0 s2 Standard 2 3 0.074 910 4 0.0318 0 s3 Standard 3 3 0.088 1082 4 0.0588 0 s4 Standard 4 3 0.107 1260 4 0.0908 0 s5 Standard 5 3 0.128 1435 4 0.1193 0 s6 Standard 6 3 0.153 1610 4 0.1503 0 -0 s7 Standard 7 3 0.290 1787 4 0.2726 0 S8 Standard 8 3 0.606 1960 4 0.4494 0 .2 s9 Standard 9 3 1.233 2135 4 0.6983 0 .3 slO Standard 10 3 1.468 23iO 4 0.7892 0 .4 d Drift 3 1.548 2484 4 0.8224 0 .5 w Wash 3 0.057 2726 4 #.#### 0 -6 u Serum blk 1 3 0.080 2839 4 0.0451 0 .7 u Serum blk 2 3 0.073 3011 4 0.0312 0 -8 u Serum spk 1 3 0.080 3189 4 0.0436 0 -9 u Serum spk 2 3 0.081 3361 4 0.0457 0 :0 u Serum spk 3 3 0.088 3537 4 0.0584 0 .1 u Serum spk 4 3 0.091 3712 4 0.0650 0 .2 u Serum spk 5 3 0.115 3887 4 0.1016 0 '3 u Serum spk 6 3 0.095 4062 4 0.0713 0 .4 u Serum spk 7 3 0.090 4236 4 0.0633 0 .5 u Serum spk 8 3 0.096 4413 4 0.0728 0 '6 d Drift 3 1.509 4587 4 0.8060 0 L7 w Wash 3 0.057 4813 4 0 '8 u F54105-24 3 0.085 4939 4 0.0541 0 29 u F54075-24 3 0.070 5111 4 0.0236 0 30 u F54081-24 3 0.068 5282 4 0.0202 0 31 u F54107-24 3 0.066 5464 4 0.0153 0 32 u F54104-24 3 0.070 5635 4 0.0236 0 33 u F54086-24 3 0.067 5814 4 0.0175 0 34 u F54074-24 3 0.075 5994 4 0.0352 0 -3 u F54085-24 3 0.055 A 6162 4 0 36 u F54083-24 3 0.055 6336 4 0 37 u F54100-24 3 0.055 6508 4 0 38 d ]Drift 3 1.472 6688 4 0.7909 0 39 w Wash 3 0.057 '6927 4 #.#### 0 40 u Blk 1 3 0.073 7038 4 0.0310' 0 41 u Blk 2 3 0.065 7210 4 0.0137 0 42 u Spk 1 3 0.078 7391 4 0.0402 0 43 u Spk 2 3 0.086 7565 4 0.0553 0 44 u Spk 3 3 0.087 7739 4 0.0578 0 u Spk 4 3 0.088 7912 4 0.0592 0 46 u Spk 5 3 0.093 8091 4 0.0674 0 47 u Spk 6 3 0.092 8263 4 0.0658 0 48 u Blk 3 3 0.078 8439 4 0.0412 0 IX9 u F54085-24 3 0.066 8615 4 0.0155 0 :,0; d Drift 3 1.506 8789 4 0.8050 0 @il w Wash 3 0.057 9028 4 #.#### 0 52 u F54083-24 3 0.068 9142 4 0.0208 0 -3 u F54100-24 3 0.066 9313 4 0.0153 0 Page 1 of 2 oool9,9 .995-05-09 15:09 .0utPut of : 950509Al Fluoride 1.5 Fluoride L ppm PPM "os Typ Ident Ch Result F Time Ch Result F Time @4 u F54077-24 3 0.067 9489 4 0.0169 0 -5 u F54094-24 3 0.065 9663 4 0.0133 0 -.6 u F54091-24 3 0.065 9842 4 0.0139 0 7;7 u F54108-24 3 0.065 10012 4 0.0135 0 -18 u F54102-24 3 0.063 10188 4 0.0090 0 :@9 u F54097-24 3 0.070 10364 4 0.0242 0 '0 u F54071-24 3 0.064 10540 4 0.0121 0 )l u F54093-24 3 0.064 10716 4 0.0106 0 '.)2 d Drift 3 1.493 10890 4 0.7997 0 @1:3 w Wash 3 0.057 11098 4 0 ,)4 u F54095-24 3 0.068 11236 4 0.0208 0 .i u F54101-24 3 0.067 11414 4 0.0171 0 :6 u F54110-24 3 0.066 11590 4 0.0151 0 -@7 u F54096-24 3 0.064 11772 4 0.0117 0 ,8 u F54090-24 3 0.084 11943 4 0.0510 0 .19 u F54078-24 3 0.071 12115 4 0.0261 0 0u F54088-24 3 0.070 12282 4 0.0242 0 -1 u F54106-24 3 0.805 12480 4 0.5348 0 u F54089-24 3 0.435 12655 4 0.3636 0 @3 u. F54087-24 3 0.574 12830 4 0.4347 0 -,4 d Drift 3 0.223 13006 4 0.2195 0 5w Wash 3 0.057 13117 4 0 .6 u F54080-24 3 0.089 13335 4 0.0606 0 ;7 u F54092-24 3 0.084 13517 4 0.0510 0 -8 u F54109-24 3 0.077 13 693 4 0.0389 0 9u F54114-24 3 0.074 13865 4 0.0322 0 -,.0. u Blk 1 3 0.068 14042 4 0.0204 0 :,Ol u Blk 2 3 0.066 14218 4 0.0145 0 :')2 u Spk 1 3 0.105 14392 4 0.0873 0 :-3 u Spk 2 3 0.118 14568 4 0.1063 0 :'@4 u F54113-24 3 0.088 14743 4 0.0584 0 .,5 u F54082-24 3 0.079 14918 4 0.0430 0 :@6 d Drift 3 1.423 15092 4 0.7713 0 :0,7 w Wash 3 0.057 15331 4 #.#### 0 --@8 u F54111-24 3 0.088 15442 4 0.0592 0 )u F54098-24 3 0.086 15618 4 0.0549 0 @10 u F54084-24 3 0.084 15794 4 0.0514 0 :"l u F54079-24 3 0.083 15968 4 0.0500 0 )2 u F54116-24 3 0.088 16145 4 0.0598 0 )3 d Drift 3 1.457 16318 4 0.7849 0 34 w Wash 3 0.057 16558 4 0 ,it rw RunOut Wash 3 0.057 16793 4 0 Page 2 of 2 Cl@.libra--Li-cnl-inrve ol7 Fl!gorieieL order Me.3su-reii gnu! 000200 -alibra4L":Drc,u.r.ve of 950SORAi 0 Fluoride l.S nr,7cR'27 -Fe47WE f*Aieasured 000201 Raw da'.-01MF q@Sg-qD9AI Fluor'Lde 1.5 ii f Li @te peaks -Z'L- , Not ii z f 00020*-" /r. Lri .......................... . .......... rr IX! .......-.-. ---- .............. tJ .... ............ -LIOZO()O IL 4c e.D 1 -EOrE . ......... e ep t,., . ................. !ILI . .......... . .................................... ................ . . ........ . ....... ----------- ----- ---- ----------------............ .995-05-09 15:59 output of 9505O9Bl oftware version 6.1 cl990,93 perator DDW ate of the Analysis 1995-05-09 15:08 inalysis File Name C:\SKALAR\DATA\HWIDATA\SERUM\950509Bl 'luoride 1.5 'alibration original time-date stamp : 1995-05-09 09:48 .alibration order = Inverse Logarithm .,lope s x - cl @esult = 101 s 1 x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode t2 = tl = 10 = -0.00000 0.00061 -1.24329 'luoride L 'alibration original time-date stamp 1995-05-09 09:48 'alibration order = 2 7orrelation r = 0.99974 lesult = a2 X2 + al * x + aO 12 = .il= 10 = 0.00000 0.00020 -0.00554 3ampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SA1000 :1 : 50 sec. : 120 sec. : I sec. : Single : None : 70 mm. )iluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample :1 Dilution runs : 1 .User file : TXT Reproces : No 00020C) .995-05-09 15:59 output of 950509Bl 'li.iorid1e.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 Debubbled Yes :0 : No : No : 4095 :0 : Off sl standard : Ignore s2 standard : Ignore s3 standard : Ignore s4 standard : Ignore s5 standard : Ignore s6 standard : 0.150 s7 standard : 0.300 S8 standard : 0.600 S9 standard : 1.200 slO standard : 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula output .?lljorideL Path number Signal type Decolor system Number dilute Resample. dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off 000207 .995-05-09 15:59 OutPut of sl standard : 0.015 s2 standard : 0.030 s3 standard : 0.060 s4 standard : s5 standard : 0.090 0.120 s6 standard : 0.150 s7 standard : Ignore ss standard : Ignore S9 standard : Ignore slO standard : Ignore order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 s6c eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula c4:=c3 output 950509BI o 995-05-09 15:59 OutPut of 9505O9Bl Fluoride 1.5 Fluoride L PPM PPM 'os Typ Ident Ch Result F Time Ch Result F Time :t iw Initial Wash 3 0.057 65 4 0 t Tracer 3 1.509 212 4 0.8062 0 d Drift 3 1.520 386 4 0.8106 0 w Wash 3 0.057 651 4 #.#### 0 u F54106-24 3 0.080 745 4 0.0438 0 u F54089-24 3 0.083 944 4 0.0494 0 u F54087-24 3 0.078 1093 4 O@0406 0 d Drift. 3 1.619 1262 4 0.8540 0 w Wash 3 0.057 l@03 4 0 :.-t rw Run out wash 3 0.057 1737 4 0 Page 1 of .995-05-09 15:44 output of : 9505O9Bl Fluoride 1.5 Fluoride L PPM PPM 5os Typ Ident Ch Result F Time Ch Result F Time it iw Initial Wash 3 0.057 65 4 #.#### 0 t Tracer 3 1.490 212 4 0.7985 0 1d Drift 3 1.482 386 4 0.7951 0 iw Wash 3 0.057 551 4 0 u F54106-24 3 0.113 751 4 0.0989 0 u F54089-24 3 0.159 926 4 0.1577 0 u F54087-24 3 0.108 1101 4 0.0916 0 d Drift 3 1.609 1262 4 0.8493 0 w Wash 3 0.057 1503 4 0 it rw Run out wash 3 0.057 1737 4 0 Page 1 of AiL Air* 4-L'P'O()O S "E-=,kCf@ I PM.= T 995-05-11 13:19 output of 95051IA1 oftware version 6.1 cl990,93 perator DDW ate of the Analysis 1995-05-11 08:16 nalysis File Name C:\SKALAR\DATA\HWIDATA\SERUM\9505llAl luoride 1.5 alibration order = Inverse Logarithm 'lope x cl @- esult = 101 s I x =,corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode 2= .1 = .0 = -0.00000 0.00060 -1.24775 'luoride L 'alibration order = 2 7orrelation r = 0.99962 @esult = a2 X2 + al * x + aO 12 = -1 = .0 = 0.00000 0.00019 -0.00028 @'ampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SA1000 :1 : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. )iluter needle Height dilution Factor dilution Volume Resample Dilution runs : 80 : 10 : 2.5 :1 :1 rilm nil. User file : TXT Reproces : No 447C)R S.I 00021L,--" ^195-05-11 13:19 output of 95051IA1 Iiioride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 : Debubbled : Yes :0 : No : NO : 4095 :0 ; Off sl standard : Ignore s2 standard : Ignore s3 standard : Ignore s4 standard : Ignore s5 standard : Ignore s6 standard : 0.150 s7 standard : 0.300 S8 standard : 0.600 S9 standard : slO standard : 1.200 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula output 'luoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off oooZIL-3 .995-05-11 13:19 output of si standard : 0.015 s2 standard : 0.030 s3 standard : 0.060 s4 standard : 0.090 s5 standard : 0.120 s6 standard : 0.150 s7 standard : Ignore S8 standard : Ignore S9 standard : Ignore slO standard : Ignore Order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter . No Regeneration No formula c4:=c3 output 950511AI 0002111- -995-05-11 13:19 OutPut of 950511AI Fluoride 1.5 Fluoride L PPM PPM los Typ Ident Ch Result F Time Ch Result F Time it iw Initial Wash 3 0.057 65 4 0 t Tracer 3 1.472 208 4 0.7342 0 d Drift 3 1.472 382 4 0.7342 0 iw Wash 3 0.057 624 4 0 sl Standard 1 3 0.064 729 4 0.0164 0 s2 Standard 2 3 0.070 913 4 0.0287 0 s3 Standard 3 3 0.085 1085 4 0.0586 0 7 s4 Standard 4 3 0.107 1260 4 0.0921 0 s5 Standard 5 3 0.127 1435 4 0.1194 0 s6 Standard 6 3 0.153 1610 4 0.1498 0 0 s7 Standard 7 3 0.289 1784 4 0.2642 0 -- S8 Standard 8 3 0.607 1959 4 0.4285 0 ",2 s9 Standard 9 3 1.233 2133 4 0.6527 0 3 slO Standard 10 3 1.468 2309 4 0.7329 0 -4 d Drift 3 1.509 2484 4 0.7476 0 -5 w Wash 3 0.057 2725 4 #.#### 0 -6 u F54113-48 3 0.094 2836 4 0.0722 0 -7 u F54082-48 3 0.091 3011 4 0.0685 0 -3 u F54111-48 3 0.096 3185 4 0.0758 0 I-9 u F54098-48 3 0.091 3359 4 0.0685' 0 10 u F54084-48 3 0.103 3537 4 0.0863 0 -1 u F54079-48 3 0.082 3711 4 0.0523 0 .2 u F54116-48 3 0.101 3887 4 0.0844 0 '@@3 u F54113-192 3 0.097 4061 4 0.0775 0 @z4 u F54082-192 3 0.089 4237 4 0.0639 0 '5 u F54111-192 3 0.081 4413 4 0.0512 0 ,1 .6 d Drift 3 1.435 4585 4 0.7213 0 '-7 w Wash 3 0.057 4732 4 0 ..;8 u F54098-192 3 0.088 4936 4 0.0626 0 u F54079-192 3 0.089 5111 4 0.0639 0 .!o ii F54084-192 3 0.094 5286 4 0.0729 0 u F5411@6-192 3 0.085 5458 4 0.0573 0 u F54113-360 3 0.085 5638 4 0.0586 0 u F54082-360 3 0.073 5810 4 0.0353 0 @4 u F54111-360 3 0.087 5984 4 0.0611 0 u F54098-360 3 0.082 6164 4 0.0519 0 36 u F54079-360 3 0.091 6336 4 0.0681 0 ';7 u F54084-360 3 0.092 6513 4 0.0699 0 -.3 d Drift 3 1.481 6686 4 0.7374 0 :i9 w Wash 3 0.057 6926 4 0 -0 u F54116-360 3 0.109 7039 4 0.0959 0 'l u BLK 1 3 0.065 7213 4 0.0188 0 -,:2 u SPK 1 3 0.094 7387 4 0.0727 0 3u SPK 2 3 0.112 7565 4 0.1002 0 -:4 u F54082-D22 3 0.085 7739 4 0.0586 0 'IT5 u F54111-D22 3 0.084 7912 4 0.0569 0 46 u F54079-D22 3 0.083 8085 4 0.0536 0 !7 u F54116-D22 3 0.083 8263 4 0.0540 0 -18 u F54082-D28 3 0.087 8438 4 0.0607 0 49 u F54111-D28 3 0.078 8614 4 0.0447 0 'IzO d Drift 3 1.519 8787 4 0.7510 0 51 w Wash 3 0.057 9027 4 #.#### 0 712 u F54079-D28 3 0.077 9i38 4 0.0431 0 .3 u F54116-D28 3 0.08'L 9314 4 0.0502 0 Page 1 of 2 ooo2l@:, .995-05-11 13:19 output of 9505IIAl Fluoride 1.5 Fluoride L PPM PPM -os Typ Ident Ch Result F Time Ch Result F Time 4u F54099-4 3 0.071 9490 4 0.0317 0 5u F54099-8 3 0.070 9662 4 0.0298 0 6u F54099-1'2 3 0.105 9840 4 0.0894 0 7u F54099-24 3 0.118 10015 4 0.1075 0 3 11 F54099-48 3 0.084 10190 4 0.0569 0 9u F54099-192 3 0.074 10362 4 0.0371 0 0u F54099-360 3 0.070 10538 4 0.0306 0 1u p&4999.-96i a &-.-65 t Pz 107t5 4 0 . #-I@#rp# 6 2d Drift 3 1.504 10888 4 0.7457 0 w Wash 3 0.057 11129 4 0 )4 u F54112-4 3 0.082 11237 4 0.0527 0 I-) u F54112-8 3 0.094 11415 4 0.0735 0 @6 u F54112-12 3 0.100 11591 4 0.0827 0 u F54112-24 3 0.088 11765 4 0.0624 0 8u F54112-48 3 0.100 11941 4 0.0815 0 9u F54112-192 3 0.094 12115 4 0.0725 0 0u F54112-360 3 0.078 12289 4 0.0452 0 1u SPK 63-1 3 0.095 12465 4 0.0750 0 '2 u SPK 63-2 3 0.110 12641 4 0.0973 0 '3 u SPK 63-3 3 0.092 12817 4 0.0689 0 4d Drift 3 1.469 12989 4 0.7334 0 5w Wash 3 0.057 13166 4 0 u SPK 126-1 3 0.117 13342 4 0.1061 0 7u SPK 126-2 3 0.149 13518 4 0.1452 0 8u SPK 126-3 3 0.157 13692 4 0.1537 0 '9 d Drift 3 1.496 13865 4 0.7427 0 '0 w Wash 3 0.057 14107 4 0 it rw Runout Wash 3 0.057 14340 4 0 Page 2 of 2 00021i*@ ,?,TZ,OOO l@al --;7 ooo2lL,'@ -----.-.-.-...................... . . ................. .......... ............. ....... .......... . ......... .......... ............ ............... . .................. ........... ............. ------ ..........-. - ------.......... .. ...................................7. ........................ al txl . ................ . ............. ........... ,ZI_T_ C.T 13P 46 43 T ihe ujz lit 3 6;3 0(.)02;4() ............................ ............... ---------- LJL- 13 In ......... - ------------- tri .......... . ....... ................. n=_@dwa4L-ofc@395n-Si@'J.94. Fliuori.d'et.5 -24 T ooo:z:.A,.A 99f,-Of,.24 OutPut of : 950524AI o@-:tware va-rsior. 6.1 -@1990,93 ,pc-rator DDW ai@-eof the Analysis 1995-05-24 li.1:39 ..nalvsis File Name C:\SKALAR\DATA\HWIDATA\SERUM\950524,Nl '. luoride 1.5 alibration order = Inverse Logarithm 31ope s x cl ',esult= 101 . s I x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode @2 = il = ;0 = -0.00000 0.00064 -1.22588 luoride L alibration order = 2 -or.relation @esult = a2 r = 0.99786 X2 + al * x + aO t2 = tl = to = -0.00000 0.00025 -0.00699 @ampler Type Number Sample Time Wash Air Time Time Take up special needle Height : SA1000 :1 : 50 sec. : 120 sec. : I sec. : Single : None : 70 mm. l,iluter needle Height dilution Factor dilution Volume Resample Diluti-on runs 80 10 2.5 1 I mm mi. User file : TXT Reproces : No 995-05-24 16:01 OutPut of 950524Al luoride 1.5 Path number Signal type Decolor svstem Number dilut--e Resarnple dil Threshold diG output Window event 3 : Debubbled : Yes :0 : No : No : 4095 :0 : Off sl standard : s2 standard : s3 standard : S4 standard : s5 standard : S6 standard : s7 standard : ss standard : S9 standard : SIO standard : Order : Inverse Dimension : PPM start Value trigger Limit Peak shape start ignore eNd ignore Measure window Filter Regt-neration Ignore Ignore Ignore Ignore Ignore 0.150 0.300 0.600 1.200 1.500 Logarithm : 500 DU : 1800 Sec : Pointed : 60 Sec : 120 Sec : 75 % : No : No formula OUtDUt ,'l-uorideL Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off 0002L>@.1 995-05-24 16:01 OutPut of sl standard : 0.015 s2 standard : 0.030 s3 standard : 0.060 s4 standard : 0.090 s5 standard : 0.120 s6 sta-,idard : 0.150 s7 standard : Ignore S8 standard : Ignore S9 standard : ignore slO standard : Ignore order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 Filter : No Regeneration : No formula c4:=c3 output 950524Al 995-05=24 1.6:01 OutPu-t of : 950524Al Fluoride 1.5 Fluoride L PPM PPM os Typ Ident Ch Result F Time Ch Result F Time iw Initial Wash 3 0.059 65 4 #.#### 0 t Tracer 3 1.452 215 4 0.4057 0 d Drift 3 1.469 389 4 0.4054 0 w Wash 3 0.059 621 4 0 sl Standard 1 3 0.069 740 4 0.0180 0 s2 Standard 2 3 0.073 916 4 0.0276 0 s3 Standard 3 3 0.087 1090 4 0.0569 0 s4 Standard 4 3 0.109 1269 4 0.0945 0 s5 Standard 5 3 0.127 1442 4 0.1182 0 s6 Standard 6 3 0.155 1618 4 0.1498 0 0 s7 Standard 7 3 0.284 1790 4 0.2417 0 - S8 Standard 8 3 0.612 1965 4 0.3431 0 2 s9 Standard 9 3 1.242 2140 4 0.4037 0 3 slO Standard 10 3 1.458 2314 4 0.4056 0 4d Drift 3 1.442 2490 4 0.4058 0 5w Wash 3 0.059 2728 4 #.#### 0 6u BLK 1 3 0.075 2841 4 0.0330 0 7u BLK 2 3 0.068 3015 4 0.0168 0 8u SPK 63-1 3 0.103 3191 4 0.0843 0 9u SPK 63-2 3 0.098 3365 4 0.0761 0 0u SPK 63-3 3 0.098 3539 4 0.0768 0 1u SPK 63-4 3 0.093 3715 4 0.0683 0 2u SPK 63-5 3 0.104 3891 4 0.0860 0 3u SPK 63-6 3 0.102 4067 4 0.0829 0 4u SPK 63-7 3 0.101 4243 4 0.0821 0 .5 u BLK 3 3 0.072 4415 4 0.0264 0 .6 d Drift 3 1.455 4591 4 0.4056 0 ,7 w Wash 3 0.059 4831 4 #.#### 0 .8 u BLK 4 3 0.073 4939 4 0.0273 0 @9 u F54088-48 3 0.068 5118 4 0.0156 0 0u F54099-48 3 0.071 5290 4 0.0233 0 1u F54089-48 3 0.066 5466 4 0.0110 0 @;2 u F54087-48 3 0.069 5644 4 0.0192 0 s3 u F54080-43 3 0.067 5812 4 0.0141 0 u F54092-48 3 0.066 5988 4 0.0095 0 u F54109-48 3 0.070 6168 4 0.0216 0 .6 u F54114-48 3 0.071 6344 4 0.0228 0 .7 U F54088-DY8 3 0.068 6517 4 0.0158 0 @3 d Drift 3 1.485 6692 4 0.4052 0 ig w Wash 3 0.059 6930 4 #.#### 0 1:0 u F54099-DY8 3 0.069 7041 4 0.0170 0 Tl u SPK 63-8 3 0.101 7218 4 0.0812 0 ,2 u SPK 63-9 3 0.099 7393 4 0.0774 0 'T3 u SPK 63-10 3 0.100 7568 4 0.0790 0 '4 u BLK 1 3 0.073 7741 4 0.0276 0 ,5 u BLK 2 3 0.065 7915 4 0.0083 0 ..r, 11 SPK 63-1 3 0.088 8093 4 0.0597 0 17 u SPK 63-2 3 0.098 8267 4 0.0765 0 '8 U SPK 63-3 -1 0.102 8443 4 0.0838 0 -L9 u F54089-DY8 3 0.072 8t:-)19 4 0.0247 0 0d Drift 3 1.471 8793 4 0.4054 0 w Wash 3 0.059 9028 4 #.#### 0 -)2 u F54087-DY8 3 0.075 9143 4 0.0328 0 u F54080-DYS 3 0.075 9319 4 0.0323 0' Page 1 of 2 00022(". 995-05-24 16:01 OutPut of : 950524Al Fluoride 1.5 Fluoride L PPM PPM os Typ Ident Ch Result F Time Ch Result F Time 4 11 F54C)92-DY8 3 0.077 9493 4 0.0365 0 5u F54109-DY3 3 0.073 9667 4 0.0280 0 u F54114-DY8 3 0.077 9843 4 0.0365 0 '7 1 u F54088-DI5 3 0.061 10022 4 #.#### 0 3u F54099-DI5 3 0.073 10194 4 0.0266 0 9u F54089-Dl5 3 0.068 10370 4 0.0165 0 0u F54087-Dl5 3 0.069 10542 4 0.0175 0 1u F54080-Dl5 3 0.066 10718 4, 0.0107 0 2d Drift 3 1.469 10893 4 0.4054 0 3w Wash 3 0.059 11133 4 #.#### 0 4u F54092-Dl5 3 0.070 11244 4 0.0216 0 f, F54109-Dl5 3 0.072 11418 4 0.0249 0 u F54114-Dl5 3 0.068 11594 4 0.0146 0 7u F54089-D22 3 0.068 11767 4 0.0158 0 3u F54087-D22 3 0.067 11944 4 0.0134 0 9u F54109-D22 3 0.068 12118 4 0.0146 0 0u F54114-D22 3 0.069 12296 4 0.0170 0 1u F54089-D28 3 0.066 12466 4 0.0114 0 2u F54087-D28 3 0.068 12642 4 0.0168 0 3u F54109-D28 3 0.069 12822 4 0.0170 0 4d Drift 3 1.455 12994 4 0.4056 0 5w Wash 3 0.059 13222 4 #.#### 0 6u F54114-D28 3 0.069 13345 4 0.0182 0 7u SPK 63-4 3 0.078 13515 4 0.0379 0 3u SPK 63-5 3 0.089 13695 4 0.0610 0 9u SPK 63-6 3 0.098 13872 4 0.0763 0 0u SPK 63-7 3 0.097 14045 4 0.0750 0 u F54105-48HR 3 0.069 14224 4 0.0180 0 2u F54075-48HR 3 0.072 14393 4 0.0261 0 .3 d Drift 3 1.411 14569 4 0.4060 0 ,4 w Wash 3 0.059 14810 4 0 .ft rw Runout Wash 3 0.059 15044 4 0 Page 2 of 2 00022e ............. ............ .............. .......... ..... r ........... ................ .......... ...................... ...... .. ........ ........... rli ........................ ................ . ............................................ ................. .... A N d..31-. ir@!--.1 ::tq.p ;@:ii 1-..p .. ...... . ..... r.:n . .............. ---------- ............................ .. ............... ........... . ........ - --------- ............... .......... . ......... .......... ................. .......... . ...................... ............ . ..-.-.-.-.-.-.-..-..-.-.-................. .............. ........... .................. ..................... .......................... .................... -------------------- ........................ ................. ............................................ ............... .......... ............. ........ . 7.k@ I 41 Pip/ 7z 73 ........ . ........... . .. ............... . ........................ ........... ............... ....... .. ........... 16, .......... ............... . ..... .. .......... .................... . .. ....... ......... .............. HAZLCECCN W IS C 0 N S IN POST OFFICE O@-@ 7545 MAC)ISON, VVI 07-7545 Sponsor: 3M St. Paul, Minnesota FINAL REPORT a CORNING Company E-1 I/-- sp K,ooxIC33OMiM C@o 0 /co@,o- Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6246 in Rabbits Author: Steven M. Glaza Study Completion Date: September 14, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-159 P h o n e 6 0 8 - -11 - 4 4 7 1 EXP R ESS-NIAI L D E L IV E R Y Page I of 38 3301 K IN S M A N BLVD. MADI Fax SON, 6 0 8 -2 4 1 - 7 2 2 7 Wl 53704 Page 2 of 38 HWI 6329-159 QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality Assurance Unit of Hazleton Wisconsin, Inc., in accordancewith the Food and Drug Administration (FDA)Good LaboratoryPracticeRegulations,21 CFR 58.35 (b) (6) (7). The following inspectionswere conducted and findings reported to-the Study Director and management. Written status reports of inspections and findings are issued to Hazleton management monthly according to standard operating procedures. Inspection Dates From To Phase Date Reported to Date to Study Director Management 03/22/95 04/10/95 04/27/95 07/05/95 09/12/95 03/22/95 04/10/95 04/27/95 07/10/95 09/12/95 Protocol Review Protocol Amendment Animal Observation Data/Report Review Report Rereview 03/22/95 04/10/95 04/27/95 07/10/95 09/12/95 03/10/95 05/10/95 05/10/95 08/10/95 10/10/95 Represe@tari-"v'e@@;,'aol-itAyssurance Unit Date Page 3 of 38 STUDY IDENTIFICATION Single-Dose IntravenousPharmacokinetic Study of T-6246 in Rabbits HWI 6329-159 Test Material T-6246 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Sponsor's Representative John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Study Director Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Study Location Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, WI 53704 Study Timetable Study InitiationDate Experimental(In-life)StartDate In-lifeEnd Date Experimental Termination Date Study Completion Date March 30, 1995 April 4, 1995 May 1, 1995 September 14, 1995 September 14, 1995 Page 4 of 38 KEY PERSONNEL Acute Toxicology Steven M. Glaza Study Director Manager Francis (Bud) W. McDonald Study Coordinator Patricia Padgham In-life Supervisor Rose M. Bridge Report Supervisor Toxicology Support Kathy Myers Manager Calvin L. Horton Supervisor HWI 6329-159 Ouality Assurance Sherry R. W. Petsel Manager Laboratory-Animal Medicine Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Anatomical Pathology Thomas E. Palmer, PhD Anatomical Pathologist Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data Page 5 of 38 CONTENTS Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compliance Test and Control Materials Test System Procedures Results Discussion Signature Pathology Report Table I IndividualBody Weights (g) 2 IndividualClinical Signs 3 IndividualPathology Comments 4 IndividualAnimal Tissue Weights and Bile Volumes Appendix A Protocol Deviations Protocol TP8084.PK Protocol Amendment No. 1 HWI 6329-159 Paqe 2 3 4 6 8 8 8 .9 10 13 13 14 15 16 18 21 22 25 26 27 37 Page 6 of 38 SUMMARY HWI 6329-159 This study was done to assess the level of systemic exposure of T-6246 when administered by a single intravenous injection to rabbits. The study was conducted using four male and four female acclimated rabbits of the Hra:(NZW)SPF strain for each treatment group as follows: Group Test Material 1 (Control) Sterile water 2 (Low) T-6246 3 (Medium) T-6246 4 (High) T-6246 5 (High) T-6246 Dose Level Number of Animals (mg/kq) Males Females 0 4 4 5 4 4 10 4 4 100 4* 4 500 4* 4 One animal sacrificed after treatment due to a possible broken back and replaced with another male animal. The animals received a single intravenous injectionof the test material at the indicateddose level into the marginal ear vein of the right ear. The dose volume was 0.5 mL/kg of body weight for Groups 1 through 4 and was 0.75 mL/kg for Group 5. Two animals/sex/dose level were sacrificed on Day 15 and the remaining animals (two animals/sex/doselevel) were sacrificed on Day 28. Clinical observations were conducted predose and at approximately 0.5, 2, and 4 hours after intravenousinjection. Additional clinical observationsand twice a day mortality checks were conducted daily thereafter until the scheduled sacrifice interval (Day 15 or Day 28). Body weights were determined on Day -7 for randomization purposes, before test or control material administration(Day 1), and at the scheduledsacrifice interval (Day 15 or Day 28). A blood sample (approximately4 mL) was collected from a marginal ear vein (left ear) of the animals at 4-, 8-, 12-, 24-, and 48-hours post-injection, and on Day 8. An approximate 4-mL blood sample was also collected on Days 15 and 22 for the animals scheduled for sacrifice on Day 28. In addition, at the time of the scheduled sacrifice (Day 15 or Day 28), approximately 20 mL of blood was obtained from each animal. All samples were centrifuged, separated into serum and cellular fractions, and sent to the Sponsor. On Day 15 or 28, the animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, and exsanguinated. An abbreviated gross necropsy examination was not done, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected, weighed (volume only determined for bile), and sent frozen to the Sponsor. Page 7 of 38 HWI 6329-159 Intravenousinjectionof T-6246 did not result in any test material-related changes in body weight gain. All animals appeared clinically normal throughout the study with the exception of one Group 4 and one Group 5 male animals that were sacrificedon Days 2 and 1, respectively,due to injury (possible broken backs). These animals were replaced and the replacement animals appeared clinically normal throughout the study. Page 8 of 38 HWI 6329-159 OBJECTIVE The objective of this study was to assess the level of systemic exposure to the test material, T-6246, when administered as a single intravenous injection to rabbits. REGULATORY COMPLIANCE This study was conducted in accordance with the U.S. Food and Drug Administration's Good Laboratory Practice Regulations for Nonclinical Laboratory Studies, 21 CFR 58, with the exception that analysis of the test mixtures for concentration,homogeneity/solubility,and stabilitywas not conducted. All procedures used in this study were in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study did not unnecessarilyduplicate any previous work. TEST AND CONTROL MATERIALS Identification The test material was identified as T-6246 and described as a clear, colorless liquid. The control material was Sterile Water for Injection,USP (Abbott Laboratories, Lot No. 86-748-DM-02; Exp. April 1, 1996), and was described as a clear, colorlessliquid. Purity and Stability The Sponsor assumes responsibilityfor test material purity and stability determinations(includingunder test conditions). Analysis of the test material mixtures for concentration,homogeneity/solubility,and stability was not conducted or requested by the Sponsor. The purity and stability of the control material were considered to be adequate for the purposes of this study. Storage and Retention The test material was stored at room temperature. The control material was stored refrigerated. Any unused test material was returned to the Sponsor after completion of all in-life testing according to Hazleton Wisconsin (HWI) Standard Operating Procedure (SOP). Any remaining vehicle may be used for other testing and will not be discardedafter issuance of the final report. Page 9 of 38 Safety Precautions HWI 6329-159 The test and control material handling procedures were according to HWI SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPF strain were received from HRP, Inc., Kalamazoo, Michigan on March 8, 1995 and maintained at the Hazleton Wisconsin facility at 3301 Kinsman Boulevard, Madison, Wisconsin. Housing After receipt, the animals were acclimatedfor a period of at least 7 days. During acclimation and throughout the study, the animals were individually housed in screen-bottom stainless steel cages in temperature- and humiditycontrolled quarters. Environmentalcontrols for the animal room were set to maintain a temperature of 190 to 230C. a relative humidity of 50% 20%, and a 12-hour light/12-hourdark lighting cycle. In cases where variationsfrom these conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal Diet The animals were provided access to water ad libitum and a measured amount of Laboratory Rabbit Diet HF #5326, PM'IFeeds, Inc. The feed is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Samples of the water are periodically analyzed by HWI. There were no known contaminants in the feed or water at levels that would have interferedwith or affected the results of the study. Selection of Test Animals The animals were identified by animal number and corresponding ear tag and were placed into study groups using a stratified body weight randomization program. The randomization body weights were determined on Day -7. The weight variation of the animals for each group of each sex selected for the study did not exceed 2 standard deviations of the mean weight, and the mean body weights for each group of each sex were not statisticallydifferent at the 5%,probabilitylevel. Page 10 of 38 HWI 6329-159 Study Design Animals weighing from 2,523 to 3,276 g at initiationof treatmentwere placed into the followingstudy groups: - Group Dose Test/Control Dose Level Volume Number of Animalsa Material (mg/kq) (mL/kq) Males Females 1 (Control)Sterilewater 0 0.5 4 4 2 (Low) T-6246 5 0.5 4 4 3 (Medium) T-6246 10 0.5 4 4 4 (High) T-6246 100 0.5 4* 4 5 (High) T-6246 500 0.75 4* 4 one animal sacrificedafter treatment due to a possible broken back and replaced with another male animal. a Two animals/sex/doselevel were sacrificedon Day 15. The remaining animals (two animals/sex/doselevel) were sacrificed on Day 28. Justificationfor SpeciesSelection Historically,the New ZealandWhite albino rabbit has been the animal of choice because of the large amount of background informationon this species. PROCEDURES Dose Preparation-andAdministration The test materialwas dilutedwith sterilewater to achievea specific concentrationfor each dose level in Groups 2 to 5. An individualdose of each respectivetest solutionor controlwas calculatedfor each animal based on its body weight on the day of treatment. The respectivetest solutionwas administeredby intravenousinjectioninto the marginalear vein of the right ear over approximately22 to 60 seconds. The prepared test solutionswere stored at room temperatureuntil administered. After administration,any remaining test solutionswere discarded. Reason for Route of Administration Intravenousinjectionis an acceptableroute to assess systemicexposure. Page 11 of 38 HWI 6329-159 Observations of Animals Clinical observations were conducted predose and at approximately 0.5, 2, and 4 hours after intravenousinjection. Additionalclinical observationsand twice a day mortality checks were conducted daily thereafter until the scheduled sacrifice interval (Day 15 or Day 28). Body weights were determined on Day -7 for randomization purposes and before test or control material administration(Day 1). Additional body weights were determined at the scheduled sacrifice interval (Day 15 or Day 28) or at unscheduled sacrifices (when survival exceeded I day). Sample Collections A blood sample (approximately 4 mL) was collected from the marginal ear vein (left ear) of the animals at 4-, 8-, 12-, 24-, and 48-hours post-injection, and on Day 8. An approximate 4-mL blood sample was also collected on Days 15 and 22 for the animals scheduled for sacrifice on Day 28. In addition, at the time of necropsy, approximately 20 mL of blood was obtained from the posterior vena cava of each animal. All samples were stored at room temperature, and then centrifuged, separated into serum and cellular fractions. These samples were then stored in a freezer set to maintain a temperature of -200C 100C until shipped to the Sponsor. Pathology The animals sacrificed on Days 1 and 2 due to apparent broken backs were necropsied in the same manner as the animals surviving to the scheduled sacrifices with the exception that they received an abbreviated gross necropsy examination and any abnormalities were recorded. On Day 15, the first two animals/sex assigned to each dose level (based on the group assignment randomization) were anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the posteriorvena cava, and exsanguinated. An abbreviated gross necropsy examination was not done, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected, weighed (volume only determined for bile), and immediately placed in a freezer set to maintain a temperature of -200C 100C. After tissue/bile collection, the animals were discarded. The remaining two animals/sex/dose level were anesthetized, bled, and exsanguinated on Day 28 in the same manner as the animals sacrificed on Day 15. Page 12 of 38 Shigment of Tissues HWI 6329-159 After completionof the in-lifephase, the blood samples (serumand cellular fractions),livers,bile,and kidneyswere sent frozen (on dry ice) to the Sponsor (James D. Johnson, 3M E.E. & P.C., Bldg. 2-3E-09, 935 Bush Avenue, St. Paul, MN, 55106), along with their correspondingweights or volumes. The Sponsor is responsiblefor the retentionand dispositionof the samples.. HWI does not accept any responsibilityfor the analysisof the samples collectedin this study nor are these resultspresentedin this report. StatisticalAnalyses No statisticalanalyseswere required by the protocol. Location of Raw Data, Records, and Final Report The raw data, records,and an originalsigned copy of the final reportwill be retained in the archives of HWI in accordancewith HWI SOP. Page 13 of 38 HWI 6329-159 RESULTS Body Weights Individual body weights are in Table 1. There was no meaningful effect on body weight gain during the study. Clinical Observations Individualclinical signs are in Table 2. All animals appearednormal throughout the study with the following exceptions: One Group 4 male (No. F54106) treated with T-6246 at 100 mg/kg appeared to have injured its back on Day 2 and was sacrificed, necropsied, and replaced with No. F54099. The replacement animal appeared normal throughout the study. One Group 5 male (No. F54076) treated with T-6246 at 500 mg/kg appeared to have injured its back on the day of treatment and was sacrificed, necropsied, and replaced with No. F54112. The replacement animal appeared normal throughout the study. Pathology Individual animal pathology comments are presented in Table 3. Individual animal tissue weights and bile volumes are in Table 4. The necropsy of animal No. F54106 revealed the skeletal muscle surrounding the lumbar spinal cord was diffusely dark red. In animal No. F54076, the skeletal muscle surrounding both hind limbs and the sacral spinal cord was diffusely dark red. The remaining animals survived to their scheduled sacrifice and were not examined grossly, although tissues were saved. Page 15 contains a pathology report by the study pathologist. DISCUSSION The level of systemic exposure of T-6246 was evaluated in male and female albino rabbits when administeredas a single intravenous injection at levels of 5, 10, 100, and 500 mg/kg. There were no test material-relatedeffects in any of the animals following administrationof this material. Page 14 of 38 SIGNATURE HWI 6329-159 Nk@\ . @@ ff- Steven M. Glaza 6 Study Director Acute Toxicology CL - ILk-1@ 7@@ Date Page 15 of 38 HWI 6329-159 PATHOLOGY REPORT There were two male rabbits sacrificed on Day 1 or Day 2 and necropsied. At necropsy, the skeletal muscle surrounding the lumbar spinal cord in Animal No. F54106 (100 mg/kg of body weight) was diffusely dark red. In Animal No. F54076 (500 mg/kg of body weight), the skeletal muscle surrounding both hind limbs and the sacral spinal cord was diffusely dark red. The findings in these animals are indicative of injury of undeterminedetiology to the caudal region of the back. The liver, bile, and both kidneys from these animals were collected as required by protocol. After necropsy, the animals were discarded. -Th@om@as4t.' P-al-mer,-44@DPathologist (6329-159.lhm) 051295 Date Page 16 of 38 HWI 6329-159 Table 1 IndividualBody Weights (g) Random- Animal ization Initial Terminal Sex Number (Day -7) (Day 1) Day 15 Day 28 Group 1 (Control)- SterileWater for Injection (0 mg/kq) Male Female F54105 F54075 F54081 F54107 F54104 F54086 F54074 F54085 2,603 2,531 2,373 2,544 2,665 2,526 2,540 2,553 2,761 2,539 2,526 2,731 2,890 2,632 2,703 2,667 2,933 2,703 - 3,130 2,775 - 2,874 3,056 2,990 3,014 Male Female F54083 F54100 F54077 F54094 F54091 F54108 F54102 F54097 Group 2 2,381 2,675 2,459 2,466 2,717 2,412 2,690 2,439 T-6246 (5 mg/kq) 2,598 2,869 2,592 2,601 2,538 3,030 - - 2,785 2,622 2,839 2,533 2,996 2,790 - - 2,859 2,916 3,290 2,928 Male Female F54071 F54093 F54095 F54110 F54096 F54090 F54078 Group 3 T-6246 (10 mg/kq) 2,487 2,652 2,662 2,622 2,523 2,776 2,772 2,642 2,709 2,993 - 2,719 2,772 2,542 2,710 2,802 2,914 2,740 2,842 2,959 3,022 - 3,126 3,162 3,022 3,244 Not required. -Sex Male Female Page 17 of 38 HWI 6329-159 Table I (Continued) Individual Body Weights (g) Animal Number Randomization (Day -7) Initial (Day 1) Terminal Day 15 Day 28 Group 4 - T-6246 (100 mg/kq) F54088 F54106 F54089 F54087 F54099 2,494 2,677 2,597 2,578 2,360 2,582 2,833 2,662 2,665 2,764 2,771 2,803 (2) - 3,004 - 2,911 3,060 - F54080 F54092 F54109 F54114 2,498 2,400 2,592 2,776 2,622 2,540 2,657 2,933 2,835 2,667 - 2,979 3,296 Male Female Group 5 T-6246 (500 mg/kq) F54076' F54113 F54082 F54111 F54112 2,655 2,667 2,734 2,547 2,996 2,678 2,753 2,843 2,784 3,276 - 2,896 - 3,530 F54098 F54084 F54079 F54116 2,565 2,510 2,468 2,442 2,679 2,600 2,526, 2,611 2,856 2,821 - 3,216 3,228 - 2,959 3,002 - Not required. a Animal No. F54106 was originally selected by the randomization program.for use in the study and was treated. This animal was sacrificed on Day 2 due to a broken back and was replaced with No. F54099. Number in superscripted parentheses indicates the day the body weight was taken. b Animal No. F54076 was originally selected by the randomization program for use in the study and was treated. This animal was sacrificed on Day 1 due to a broken back and was replaced with No. F54112. Sex Male Female Male Female Page 18 of 38 HWI 6329-159 Table 2 IndividualClinical Signs Animal Number Observation Hour (Day 1) 0.5 2 4 Day 2 3 to 15 16 to 28 Group I (Control)- SterileWater for Injection (0 mg/kq) F54105 Appeared normal F54075 Appeared normal It t I/ F54081 Appeared normal F54107 Appeared normal 1( 0( t F54104 Appeared normal t 1( F54086 Appeared normal t 1( It F54074 Appeared normal 1( F54085 Appeared normal Group 2 - T-6246 (5 mg/kq) F54083 Appeared normal F54100 Appeared normal F54077 Appeared normal F54094 Appeared normal F54091 Appeared normal F54108 Appeared normal F54102 Appeared normal i F54097 Appeared normal t Animal sacrificed on Day 15. Condition existed. Sex Male Female Male Female Page 19 of 38 Table 2 (Continued) IndividualClinical Signs HWI 6329-159 Animal Number Observation Hour (Day 1) 0.5 2 4 2 Group 3 - T-6246 (10 mg/kq) Day 3 to 15 16 to 28 F54071 Appeared normal F54093 Appeared normal 1( of 1( F54095 Appeared normal t It F54101 Appeared normal It It 1( F54110 Appeared normal It it F54096 Appeared normal F54090 Appeared normal F54078 Appeared normal Group 4 - T-6246 (100 mg/kq) F54088 Appeared normal F54106 Appeared normal t It Broken back Moribund sacrifice F54089 Appeared normal .1 1( t 1( It t F54087 Appeared normal It F5409 ga Appeared normal V, F54080 Appeared normal F54092 Appeared normal F54109 Appeared normal F54114 Appeared normal vi Animal sacrificed on Day 15. Condition existed. Condition not evident. a Replacement animal for Animal No. F54106. Sex Male Female Animal Number F54076 F54113 F54082 F54111 F54112 F54098 F54084 F54079 F54116 Page 20 of 38 Table 2 (Continued) IndividualClinical Signs Observation Hour (Day 1) 0.5 2 4 2 Group 5 - T-6246 (500 mg/kq) Appeared normal Broken back t Moribund sacrifice Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal vi V/ Appeared normal Appeared normal HWI 6329-159 Day 3 to 15 16 to 28 Animal sacrificed on Day 15. Condition existed. Condition not evident. t Condition evident at time of 12-hour bleeding interval. a Replacement animal for Animal No. F54076. Page 21 of 38 Table 3 Individual Pathology Comments HWI 6329-159 Animal Test Day Number Sex Died Sacrificed Necropsy Observation Group 4 T-6246 (100 mg/kq) F54106 m 2 The skeletal muscle surrounding the lumbar spinal cord is diffusely dark red. F54076 m Group 5 T-6246 (500 mg/kq) 1 The skeletal muscle surrounding both hind limbs and sacral spinal cord is diffuselydark red. Page 22 of 38 HWI 6329-159 Table 4 IndividualAnimal Tissue Weights and Bile Volumes -Sex Animal Number Sacrifice Day Weight (g) Liver Kidneys Bile Volume (mL) Group 1 (Control)- SterileWater for Injection 0 mg/kq) Male F54105 15 86.35 14.92 0.6 F54075 15 60.25 15.97 0.1 F54081 28 80.597 14.764 1.2 F54107 28 80.649 15.620 0.4 Female F54104 15 98.40 16.03 0.7 F54086 15 77.30 15.23 0.7 F54074 28 82.895 18.793 1.6 F54085 28 80.950 14.925 1.3 Group 2 T-6246 (5 mg/kg) Male F54083 15 73.15 14.87 0.7 F54100 15 77.90 17.62 0.7 F54077 28 75.699 17.864 0.3 F54094 28 78.689 17.213 0.8 Female F54091 15 84.90 14.92 1.6 F54108 15 79.90 15.40 0.4 F54102 28 101.494 18.308 1.7 F54097 28 73.621 16.214 1.4 -Sex Male Female Page 23 of 38 HWI 6329-159 Table 4 (Continued) IndividualAnimal Tissue Weights and Bile Volumes Animal Number Sacrifice Day Weight (g) Liver Kidneys Bile Volume (mL) Group 3 - T-6246 (10 mg/kq) F54071 15 77.10 15.88 0.5 F54093 15 81.40 15.50 0.8 F54095 28 85.504 15.561 1.2 F54101 28 85.668 16.120 1.0 F54110 15 83.68 15.79 1.4 F54096 15 78.10 18.70 1.1 F54090 28 76.220 15.878 2.1 F54078 28 83.460 18.130 1.0 Group 4 T-6246 (100 mg/kg) Male F54088 15 84.26 16.98 0.4 F54106 2 82.301 16.550 2.0 F54089 28 71.796 16.434 1.0 F54087 28 83.710 18.591 0.6 F54099a 15 82.143 16.038 0.5 Female F54080 15 75.42 18.32 1.2 F54092 15 78.36 16.68 F54109 28 73.958 16.915 1.6 F54114 28 80.966 19.404 2.0 a Replacement animal for Animal No. F54106. This animal had no gallbladder,so no bile was collected. -Sex Male Female Page 24 of 38 HWI 6329-159 Table 4 (Continued) Individual Animal Tissue Weights and Bile Volumes Animal Number Sacrifice Day Weight (g) Liver Kidnevs Bile Volume (mL) Group 5 - T-6246 (500 mg/kq) F54076 1 78.058 14.695 2.0 F54113 15 91.52 16.57 1.0 F54082 28 88.965 19.431 1.6 F54111 28 83.823 19.405 1.5 F54112a 15 97.824 21.069 1.4 F54098 15 78.95 14.72 1.2 F54084 15 66.42 13.18 0.8 F54079 28 83.117 14.785 1.8 F54116 28 71.962 15.793 2.0 a Replacement animal for Animal No. F54076. Page 25 of 38 APPENDIX A Protocol Deviations Protocol TP8084.PK Protocol Amendment No. I 1-1t HWI 6329-159 Page 26 of 38 Protocol Deviations HWI 6329-159 Protocol Page 6, 7. ExperimentalDesign, C. Dosing Procedures,(1) Dosing Route. Intravenous injectioninto the marginalear vein of the right ear over approximately30 to 60 seconds. Actual Procedure The rate of injectionfor one Group 5 female (No. F54098) was 22 seconds. Also, the time for dose administration for one male animal (Group 4, No. F54087)was incorrectly recorded thus the exact durationof dosing can not be determined. These deviations are not consideredto have had an adverse effect an the outcome of the study. "AZLUECC:N W IS C 0 N S IN POST OFFICF BOX 71,4,1@ MADISOtI.Wi 5.'170,'754F, Page 27 of 38 a CORNING Comr),ini Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.EXT Study Title: Single-Dose Intravenous PharmacokineticStudy of T-6246 in Rabbits Date: March 30, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-159 P @io rie 6 0 8 2 4 1 -14 E X P riE S S PvlA IL D E L ERY 3301 KINSMAN BLVD F ax MADISON, 6 vv 1 -2 -7 53704 Page 28 of 38 STUDY IDENTIFICATION TP8084.EXT Page 2 Single-Dose IntravenousPharmacokineticStudy of T-6246 in Rabbits HWI No. Test Material Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date 6i29-159 T-6246 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff,PhD 3M ToxicologyService Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612)733-1962 Steven M. Glaza Hazleton Wisconsin,Inc. P.O. Box 7545 Madison, WI 53707-7545 (608)241-7292 HazletonWisconsin, Inc. 3301 Kinsman Boulevard Madison, WI 53704 April 4, 1995 May 1, 1995 June 12, 1995 Page 29 of 38 TP8084.EXT Page 3 1. Studv Single-Dose Intravenous PharmacokineticStudy in Rabbits 2. Purpose To assess the level of systemic exposure when the test material is administeredas a single intravenousinjectionto rabbits 3. Regulatory Compliance This study will be conducted in accordancewith the following Good Laboratory Practice Regulations/Standards/Guidelinewsith the exceptionthat analysisof the test materialmixtures for concentration,solubility,homogeneity,and stabilitywill not be conducted: ( ] Conduct as a Nonregulated Study CXI 21 CFR 58 (FDA) 40 CFR 160 (EPA-FIFRA) 40 CFR 792 (EPA-TSCA) C(81)30 (Final)(OECD) 59 Nohsan No. 3850 (Japanese MAFF) Notification No. 313 (Japanese MOHW) All procedures in this protocol are in compliancewith the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study does not unnecessarilyduplicate any previous work. 4. Ouality Assurance The protocol,study conduct,and the final reportwill be audited by the Quality Assurance Unit in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedures (SOPS) and policies. 5. Test Material A. Identification T-6246 B. Physical Description (To be documentedin the raw data) C. Purity and Stability The Sponsor assumesresponsibilityfor purity and stability determinations(including-undertest conditions). Samples of test material/vehiclmeixture(s)for concentration,solubility, homogeneity,and stability analyses will be taken before administrationif requested by the Sponsor. These samples (if taken) will be sent to the Sponsor after experimental termination. D. Storage Room temperature Page 30 of 38 TP8084.EXT Page 4 E. Reserve Samples Reserve sample(s)of each batch/lotof test and control materialswill be taken for this study. The test and control material reserve sampleswill be stored at HWI in a freezer set to maintain a temperature of -20*C 10*C for 10 years per HWI SOP. The,Sponsorwill be contacted after 10 years for disposition in accordancewith the appropriate regulatory Good Laboratory Practices. F. Retention Any unused test material will be returned to the Sponsor after completionof the in-lifephase of the study. G. Safety Precautions As required by HWI SOPs and policies 6. Control Material A. Identification Sterilewater for injection B. Physical Descrigtion Clear, colorlessliquid C. Purity and Stability The purity and stabilityof this USP grade material is consideredadequate for the purposes of this study. D. Storage Refrigerated E. Reserve Samples See Section 5. E. Reserve Samples F. Retention Any remaining control material may be used for other testing and will not be discardedafter issuanceof the final report. G. Safety Precautions As required by WI SOPs and policies 7. ExperimentalDesign A. Animals (1) Species Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP, Inc. Page 31 of 38 (3) Age at Initiation Adult TP8084.EXT Page 5 (4) Weight at Initiation 2.5 to 3.5 kg (5) Number and Sex 20 males and 20 females (6) Identification Individual numbered ear tag (7) Husbandry (a) Housing Individually,in screen-bottomstainlesssteel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds, Inc.). The food is routinelyanalyzedby the manufacturer for nutritionalcomponents and environmental contaminants. (c)Water Ad libitum from an automatic system. Samples of the water are analyzed by HWI for total dissolvedsolids, specifiedmicrobiologicalcontent, selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants in the food or water that would interferewith this study. (e) Environment Environmentalcontrols for the animal room will be set to maintain a temperature of 19'C to 23*C, a relative humidity of 50% +20%, and a 12-hour. light/12-hourdark cycle. (f) Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to HWI SOPS. An adequate number of extra animals will be purchased so that no animal in obviouslypoor health is placed on test. The animals will be placed into study groups using a stratified body weight randomizationprogramwithin nine days of study initiation. Page 32 of 38 TP8084.EXT Page 6 (9) Justificationfor Species Selection Historically,the New Zealand White albino rabbit has been the animal of choice because of the large amount of background informationon this species. B. Dose Administration (1) Test Groups Group Test/Control Material- Dose Level a (mg/kq) Number of Animalsb Females 1 (Control) Sterile water 0 4 4 2 (Low) T-6246 5 4 4 3 (Medium) T-6246 10 4 4 4 (High) T-6246 100 4 4 5 (High) T-6246 500 4 4 a The dose volume will be 0.5 mL/kg of body weight. b Two animals/sex/dose level will be sacrificed on Day 15. The remaining animals (two animals/sex/dose level) will be sacrificed on Day 28. C. Dosing Procedures (1) Dosing Route Intravenous injection into the marginal ear vein of the right ear over approximately 30 to 60 seconds. (2) Reason for Dosing Route Intravenous injection is an acceptable route to assess systemic exposure. (3) Dosing Duration Single dose (4) Dose Preparation The day of treatment will be designated as Day 1. The Group 1 animals will be treated with sterile water at a dose volume of 0.5 mL/kg. The test material will be diluted with sterile water to achieve a specific concentration for each dose level in Groups 2-5. Individual doses will be calculated based on the animal's body weight taken just before test material administration. The prepared test mixtures will be stored at room temperature until administration. Page 33 of 38 D. Observation of Animals TP8084.EXT Page 7 (1) Clinical observations The animalswill be observedfor clinicalsigns of toxicitybeforetest or control materialadministration, at approximately0.5, 2.0, 4.0 hours post-injection (Day 1), and daily thereafterfor clinicalsigns and twice daily (a.m.and p.m.) for mortalityuntil the scheduled sacrifice interval(Day 15 or Day 28). Observationsmay be extended when directed by the Study Director. (2) Body Weights For randomization,before test or controlmaterial injection(Day 1), at the scheduledsacrificeinterval (Day 15 or Day 28), and at unscheduled death and sacrifices(when survivalexceeds 1 day) (3) Sample Collections (a) Freguency 4-, 8-, 12-, 24-, and 48-hourspost-injection,on Days 8, 15, 22, and at the scheduled sacrifice interval(Day 15 or Day 28) (b) Method of Collection/Number of Animals Blood samples (approximately4 mL) will be collected from the marginalear vein (leftear) of all animals at 4-, 8-, 12-, 24-, and 48-hourspost-injection,and on Day 8. Additionalsampleswill be collectedon Days 15 and 22 for the animals scheduled for sacrifice on Day 28. Approximately20 mL of blood (actualvolume to be documented in the raw data) will be obtained from the posterior vena cava at the time of the scheduled sacrifice (Day 15 or Day 28). Approximately20 mL of blood will be collected from moribund animals during the study,also, if possible. The samples will be stored at room temperature and then centrifuged,and the separate serum and cellular fractionsstored in a freezer set to maintain a temperature of -20*C 10*C. The separated serum and cellularfractionswill be sent frozenon dry ice to the Sponsor after experimentaltermination. The Sponsor is responsible for the retentionand dispositionof the samples. Page 34 of 38 Samples will be shipped to: TP8084.EXT Page 8 James D. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 James D. Johnson or his alternatewill be notified by telephoneat (612) 778-5294 prior to the shipment of the samples. E. Termination (1) Unscheduled Sacrifices and Deaths Any animal dying during the s dy or sacrificedin a moribund conditionwill be subjected to an abbreviated gross necropsy examination and all abnormalitieswill be recorded. Animals in a moribund conditionwill be anesthetizedwith sodium pentobarbital(via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. Tissues, as described in section 7.E. (3) Sample Collection,will be collectedfrom any animal dying during the study or sacrificedin a moribund condition. After necropsy,the animalswill be discarded. (2) Scheduled Sacrifices On Day 15, the first two animals/sexassigned to each dose level (based on the group assignmentrandomization)will be anesthetizedwith sodium pentobarbital(via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. The remaining two animals/sex/doselevel will be anesthetizedwith sodium pentobarbital(via injectionin the marginalear vein), bled via the vena cava, and exsanguinatedon Day 28. An abbreviated gross necropsy examinationwill not be done, however, tissues (as describedin section7.E. (3) SampleCollection)will be collected. (3) Sample Collection The whole liver, bile, and both kidneys from each animal will be collected,weighed (volumeonly determinedfor bile), and immediatelyplaced in a freezerset to maintain a temperatureof -200C IOOC. After sample collection, the animals will be discarded. The samples (liver,bile, and kidneys)will be sent frozen on dry ice to the Sponsor after experimentaltermination. The samples and their corresponding weights or volumes will be shippedto the person listed in Section 7.D.(3).(b). The Sponsoris responsiblefor the retention and disposition of the samples. Page 35 of 38 F. StatisticalAnalyses No statisticalanalysesare required. TP8084.EXT Page 9 8. Report A final report includingthose items listed below will be submitted. Descriptionof the test and control materials Descriptionof the test system Procedures Dates of experimental initiationand termination Descriptionof any toxic effects Gross pathologyfindings (if applicable) Gross pathology report (if applicable and requested by the Study Director) Individualanimal tissue weights and bile volumes 9. Location of Raw Data, Records-,and Final Report Originaldata, or copies thereof,will be availableat HWI to facilitateauditingthe study during its progressand before acceptanceof the final report. When the final report is completed, all originalpaper data, includingthose item listed below will be retained in the archives of HWI according to HWI SOP. Protocol and protocol amendments Dose preparation records In-life records Body weights Dose administration Observations Sample collection records Shipping records Pathology Records Study correspondence Final report (originalsignedcopy) The followingsupportingrecords will be retained at HWI but will not be archivedwith the study data. Animal receipt/acclimationrecords Water analysis records Animal room temperature and humidity records Refrigeratorand freezer temperature records Instrumentcalibration and maintenance records Page 36 of 38 PROTOCOL APPROVAL TP8084.EXT Page 10 hn L. Butenhoff, PhD Sponsor's Representative 3M Ni\ Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. ot.,@ Representatpve Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-159.protdsk2) Date Date 3 -3c)-7Y Date HAZLCECCON W W IS C 0 N S IN POST OFFICE BOX 7545 MADISON.WL 53707 7545. Page 37 of 38 a CORNING Company PROTOCOL TP8084.EXT Single-Dose Intravenous PharmacokineticStudy of T-6246 in Rabbits HWI 6329-159 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 SRonsor's Representative John L. Butenhoff, PhD Contractor Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, WI 53704 Study Director Steven M. Glaza Amendment No. I This amendment modifies the following portions of the protocol: EffectiveApril 4, 1995 1. Page 6. 7. ExperimentalDesign; B. Dose Administration;(1) Test Groups. The test materialmixture for Group 5 could not be preparedat the concentrationneeded to utilize a dose volume of 0.5 mL/kg. Modify footnote "a" in this sectionwith the followingunderlinedaddition: a The dose volume will be 0.5 mL/kg of body weight for Groups 1-4 and 0.75 mL/kq for Group 5. P h o n e 6 0 8 -2 4 1 4 4 7 1 E X P R E S S M A 1L D E L IV E R Y 3301 KINSMAN BLVD F ax MADISON, 6 0 8 -2 4 1 - 7 2 2 7 wi 53704 Page 38 of 38 Amendment No. 1 HWI 6329-159 Page 2 EffectiveApril 5, 1995 2. Page 6, 7. ExperimentalDesign; B. Dose Administration;(1) Test Groups. Animal No. F54076 (Group 5 male) and Animal No. F54106 (Group 4 male) were sacrificedon Days I and 2, respectively,due to injuries (apparent broken backs). Add the followingparagraph to this section: Due to the sacrifice on Day I of one Group 5 male (Animal No. F54076) and on Day 2 of one Group 4 male (AnimalNo. F54106)because of injuries (apparentbroken backs), replacement animals will be treated at the same dose levels in the same manner as for the initial animals in the study. The observations (clinical observations,body weights and sampld collections)and the termination of the animals (unscheduled sacrificesand deaths, scheduled sacrifices,and sample collection)will be conducted in the same manner as for the other animals in the study terminating on Day 15. PROTOCOL AMENDMENT APPROVAL 2. - John L. Butenhoff, PhD Date Sponsor's Representative 3M Toxicology Service Medical Department Steven M. Glaza u Date Study Director Acute Toxicology Hazleton Wisconsin, Inc. Repre!i-entate Date Quality Ass?rance Unit Hazleton Wisconsin, Inc. (6329-159.Aml.dsk3)